Method for opening tight junctions

ABSTRACT

A method of using RNA interference (RNAi) for the transient, reversible and controlled opening of the tight junctions of the blood brain barrier and/or the blood retinal barrier. This method may be used in the treatment of many diseases and disorders which require the opening of the blood brain barrier and/or blood retinal barrier. Such methods generally involve the use of an RNAi-inducing agent, such as siRNA, miRNA, shRNA or an RNAi-inducing vector whose presence within a cell results in production of an siRNA or shRNA, targeting tight junction proteins to open the blood brain barrier and/or blood retinal barrier.

INTRODUCTION

The present invention is directed to a method and use of RNAinterference (RNAi), using RNAi inducing agents, such as siRNA, miRNA,shRNA or an RNAi-inducing vector whose presence within a cell results inproduction of an siRNA or shRNA, targeting tight junction proteins, forthe transient, reversible and controlled opening of the tight junctionsof the blood brain barrier and/or the blood retinal barrier.

The brain is an energy-demanding organ requiring nutrients and oxygen,while at the same time, needing protection from other potentiallyharmful agents, for example, viral or bacterial particles, oranaphylatoxins (potentially destructive particles generated as aby-product of the immune system), which may be delivered by thecirculation to this sensitive tissue. For this reason, those cells whichline the walls of the fine capillaries that supply blood to the brain(the blood-brain barrier (BBB)) have evolved ‘tight junctions’, which,as the name implies, reduce the space between adjacent endothelial cellslining the fine capillaries of the microvasculature of the brain tovirtually zero, forming a tight seal. However, oxygen can still diffusefrom these cells, and other essential materials can be delivered to thebrain by special transporters located in the membranes of theendothelial cells.

Many attempts have been made either to break the blood brain barrier orto design delivery systems that enable pharmacological agents totraverse the endothelial cells of brain capillaries (Pardridge et al.,2005). However, complete breakdown of the blood-brain barrier would havedisastrous consequences for brain function. Previous attempts have beenmade without a comprehensive understanding of the structure of the tightjunctions (TJ's), without technologies capable of ablating transcriptsencoding TJ proteins and without a means of systemic delivery of suchagents to the endothelial cells of brain or retinal capillaries (Miller,2002).

Recently, genetically engineered proteins termed “Molecular Trojanhorses” have been described and purportedly cross the BBB via endogenousreceptor-mediated transport processes (Pardridge et al., 2006). In 2007,using a modified yet similar approach, targeted delivery of proteinsacross the BBB was reported using a lentivirus vector system, exploitingthe binding domain of apolipoprotein B to its receptor “low-densitylipoprotein receptor” (LDLR). This report proved feasible for thedelivery of proteins via the transcellular pathway, yet these approacheshave yet to address peptide cleavage from the engineered binding sitesupon delivery to the CNS (Spencer B J and Verma M, 2007). Transcellular,receptor-mediated, delivery of molecules across the BBB remains anexciting avenue for further research, however, there are other routesincluding the paracellular pathway which may be utilised.

In general, transport of components across endothelial cells of the BBBcan occur via three routes: a transcellular route, which may be mediatedvia special transporters as alluded to above, vesicular transport, or aparacellular route which allows for transport between neighbouringendothelial cells (Reese and Karnovsky, 1967; Edwards, 2001; Wolburg andLippoldt, 2002). Brain capillaries exhibit very low rates of fluid phasetranscytosis, and the paracellular route between individual cells at theBBB is sealed by TJs that are considerably tighter than in any othermicrovessels in the body. Therefore, TJs represent a key factor in thelow permeability properties associated with the BBB (Matter K and BaldaM S, 2003).

The TJ's associated with the BBB are composed of a complex ofintracellular and transmembrane proteins including occludin, junctionaladhesion molecule (JAM), claudins-1, -5, -12 and ZO-1, -2 and -3(Fanning et al., 1998; Zahraoui et al., 2000).

Claudins play an essential role in BBB function. Approximately 20members of the claudin family have also been described, claudins 1, -5and -12 predominating in TJ's of the BBB (Bazzoni, 2006). Claudins, likeoccludin, span endothelial cell membranes four times and interact withZO-1 via their C-terminus (Kausalya et al., 2001). Co-expression ofoccludin and claudin-1 in fibroblasts has been shown to result inco-localization of both proteins at the periphery of the cells inTJ-like strands (Furuse et al., 1998). Claudin-1 over-expression inMadin-Darby canine kidney (MDCK) cells increases Trans-cellularelectrical resistance (TER) and has been shown to reduce fluoresceinisothiocyanate (FITC)-dextran flux across the monolayer (Inai et al.,1999).

WO 02/014499 (Immunex) is directed to new members of the claudin family,claudin-19, -21 and -22. This application concerns the generation ofpolypeptide fragments targeting extracellular binding domains of theseclaudins, while also describing the production of recombinant protein.

Claudin-5 is a four trans-membrane protein, which when knocked out inthe mouse causes a size-selective loosening of the BBB to molecules ofless than 800 Da. Claudin-5 is considered to be endothelialcell-specific (Turksen K and Troy T C, 2004). Claudin 5−/− mice havebeen reported and the BBB is compromised in these animals. Through aseries of tracer molecule experiments and Magnetic Resonance Imaging(MRI), it was found that while removal of claudin-5 compromises thefunction of the barrier by allowing it to become permeable to moleculesof up to approximately 800 Da, the barrier can form, remaining intactand impervious to larger molecules, showing no evidence of bleeding oroedema (Nitta et al., 2003). However, these knockout mice had very highmortality rates and only survived for a few hours. As such, theseknockout mice cannot be used to study the physiology of the BBB andalternative models are needed.

Other systems have been developed to open tight junctions to enhancemucosal paracellular transport. For example, WO 04/003145 (Nastech) andWO 05/058362 (Nastech) both address the need to provide an alternativeadministration route to injection whilst maintaining the requiredbioavailability of an active ingredient. WO 04/003145 (Nastech) isdirected to the mucosal delivery of biologically active agents,permeabilising agents targeting claudin-5 which can reversibly enhancemucosal paracellular transport. These permeabilising agents are peptidesdirected against extracellular binding domains of claudin-5 whichmediate homotypic interaction of this protein with a similar protein onan adjacent cell. WO 05/058362 (Nastech) is directed to a method for theopening tight junctions in the nose which also comprises the mucosaladministration of a wide variety of antagonists to JAM1, Claudin-4 andoccludins.

However, despite these advances, many drugs are still ineffectivebecause they are unable to cross the BBB. Thus, much effort has beendirected toward understanding the TJs of brain capillary endothelialcells and retinal cells in order to identify molecular mechanisms thatcould be manipulated to enhance drug delivery across the BBB.

The controlled opening of the BBB, if achieved, would provide an avenuefor the experimental delivery of agents to the brain which was notpossible previously and could open the door to the treatment of manyconditions which involve the blood brain barrier and/or the bloodretinal barrier.

Furthermore, the controlled opening of the BBB could enable theestablishment of experimental animal models of neurodegenerative andneuropsychiatric disorders and also pave the way for controlled deliveryof therapeutic agents in a range of conditions that currently havelittle or no prospect of effective treatment, for example, agents thatmodulate neuronal function to the CNS in a range of neurodegenerativeconditions.

The present invention is directed to addressing at least some of theseproblems.

SUMMARY

According to a first general aspect of the invention, there is provideda method for the transient, reversible and size-selective opening of theblood brain barrier wherein the method comprises the use of RNAinterference (RNAi) for the transient, reversible and controlled openingof the tight junctions of the blood brain barrier and/or the bloodretinal barrier.

The silencing effect of complementary double stranded RNA was firstobserved in 1990 in petunias by Richard Joergensen and termedco-suppression. RNA silencing was subsequently identified in C. elegansby Andrew Fire and colleagues, who coined the term RNA interference(RNAi). This gene silencing phenomenon was later found to be highlyconserved in many eukaryotic cells. Thus, RNAi has been shown to beeffective in both mammalian cells and animals.

An important feature of RNAi affected by siRNA is the double strandednature of the RNA and the absence of large overhanging pieces of singlestranded RNA, although dsRNA with small overhangs and with interveningloops of RNA has been shown to effect suppression of a target gene. Inthis specification, it will be understood that in this specification theterms siRNA and RNAi are interchangeable. Furthermore, as is well-knownin this field RNAi technology may be effected by siRNA, miRNA or shRNAor other RNAi inducing agents.

Although siRNA will be referred to in general in the specification. Itwill be understood that any other RNA inducing agent may be used,including shRNA, miRNA or an RNAi-inducing vector whose presence withina cell results in production of an siRNA or shRNA targeted to a targettranscript.

RNA interference is a multistep process and is generally activated bydouble-stranded RNA (dsRNA) that is homologous in sequence to the targetgene. Introduction of long dsRNA into the cells of organisms leads tothe sequence-specific degradation of homologous gene transcripts. Thelong dsRNA molecules are metabolized to small (e.g., 21-23 nucleotide(nt)) interfering RNAs (siRNAs) by the action of an endogenousribonuclease known as Dicer.

The siRNA molecules bind to a protein complex, termed RNA-inducedsilencing complex (RISC), which contains a helicase activity and anendonuclease activity. The helicase activity unwinds the two strands ofRNA molecules, allowing the antisense strand to bind to the targeted RNAmolecule. The endonuclease activity hydrolyzes the target RNA at thesite where the antisense strand is bound. Therefore, RNAi is anantisense mechanism of action, as a single stranded (ssRNA) RNA moleculebinds to the target RNA molecule and recruits a ribonuclease thatdegrades the target RNA.

An “RNAi-inducing agent” or “RNAi molecule” is used in the invention andincludes for example, siRNA, miRNA or shRNA targeted to a targettranscript or an RNAi-inducing vector whose presence within a cellresults in production of an siRNA or shRNA targeted to a targettranscript. Such siRNA or shRNA comprises a portion of RNA that iscomplementary to a region of the target transcript. Essentially, the“RNAi-inducing agent” or “RNAi molecule” downregulates expression of thetargeted tight junction proteins via RNA interference.

Preferably, siRNA, miRNA or shRNA targeting tight junction proteins areused.

Specifically, the method involves the delivery of an effective amount ofsiRNA or shRNA targeting tight junction proteins to the subject. It willbe understood that an effective amount of the RNAi-inducing agent, suchas siRNA, is used to open the BBB to allow the passage of the activeagent treating the disorder of interest. Preferably, delivery is via asystemic route.

According to this aspect of the invention, the method results in thereversible and transient RNAi-mediated suppression of the blood brainbarrier tight junction protein transcripts in brain capillaryendothelial and/or retinal endothelial cells to allow the permeation ofmolecules, ideally less than 15 kDa, across the blood brain barrier,through the brain capillary endothelial and/or retinal endothelialcells. Opening the blood barrier to this extent will allow siRNAs whichtypically have a maximum molecular weight of approximately 10-15 kDA,preferably 13 kDa, and/or low molecular weight drugs which generallyhave a molecular weight less than approximately 2 kDA, preferablyapproximately 1 kDa or less, to cross the blood brain barrier. It willof course be understood that the blood brain barrier may be opened toallow molecules with a molecular weight greater than 15 kDa to cross theblood brain barrier.

Ideally, the method involves the systemic hydrodynamic delivery of theRNAi inducing agent, such as siRNA, miRNA or shRNA etc, to the subject.Although, non-hydrodynamic systemic delivery methods may be used.

Other delivery methods suitable for the delivery of the RNAi inducingagent (including siRNA, shRNA and miRNA etc) may also be used. Forexample, some delivery agents for the RNAi-inducing agents are selectedfrom the following non-limiting group of cationic polymers, modifiedcationic polymers, peptide molecular transporters, lipids, liposomesand/or non-cationic polymers. Viral vector delivery systems may also beused. For example, an alternative delivery route includes the directdelivery of RNAi inducing agents (including siRNA, shRNA and miRNA) andeven anti-sense RNA (asRNA) in gene constructs followed by thetransformation of cells with the resulting recombinant DNA molecules.This results in the transcription of the gene constructs encoding theRNAi inducing agent, such as siRNA, shRNA and miRNA, or even asRNA andprovides for the transient and stable expression of the RNAi inducingagent in cells and organisms. For example, such an alternative deliveryroute may involve the use of a lentiviral vector comprising a nucleotidesequence encoding a siRNA (or shRNA) which targets the tight junctionproteins. Such a lentiviral vector may be comprised within a viralparticle. Adeno-associated viruses (AAV) may also be used and the use ofthese as delivery vehicles is expanded on later.

According to a second aspect of the present invention, the delivery ofan RNAi inducing agent, preferably siRNA, miRNA or shRNA etc, accordingto the invention may be useful in the generation of an experimentalmodel for studying the action of the paracellular pathway, thephysiology of the BBB and testing the effect of drugs which cross theBBB and may previously not have been able to cross the BBB.

According to a third aspect of this invention, the present invention isapplicable for the treatment of many diseases or disorders where theblood brain barrier or blood retinal barrier is implicated.

According to a fourth aspect of this invention, there is provided apharmaceutical composition, preferably adapted for systemic delivery,comprising an RNAi inducing agent, preferably siRNA, miRNA or shRNA etc,targeting tight junction proteins to result in the reversible, transientand controlled size selective opening of the paracellular pathway of theblood brain barrier and an active agent for the treatment of a defineddisease or disorder. Ideally, the active agent is a biologically active,therapeutic agent.

DETAILED DESCRIPTION

In this specification, the term “blood brain barrier” or BBB has beenused to cover both the blood brain barrier (BBB) and the blood retinalbarrier (BRB). As expanded on above, the blood-brain barrier (BBB)contains tight junctions (TJ's) which reduce the space between adjacentendothelial cells lining the fine capillaries of the microvasculature ofthe brain to virtually zero to enable the transport of nutrients andoxygen across the BBB, while at the same time, preventing the transportof other potentially harmful agents across the BBB. The blood-retinalbarrier (BRB) is part of the blood-ocular barrier that consists of cellsthat are joined tightly together in order to prevent certain substancesfrom entering the tissue of the retina. The blood retinal barrier hastwo components, the retinal vascular endothelium and the retinal pigmentepithelium, which also have tight junctions (TJ's). Retinal bloodvessels, which are similar to cerebral blood vessels, maintain the innerblood-ocular barrier.

Additionally, the invention disclosed in the present specificationrelates to the use of RNAi techniques in general. Ideally, anRNAi-inducing agent is used including siRNA, miRNA or shRNA targeted toa target transcript, or an RNAi-inducing vector whose presence within acell results in production of an siRNA or shRNA targeted to a targettranscript. Such siRNA or shRNA comprises a portion that iscomplementary to a region of the target transcript. Thus, it will beunderstood that RNAi can be effected using both siRNA and shRNA inparticular.

The RNAi inducing agent of the invention interferes or interrupts thetranslation of mRNA. Such RNAi inducing agents can be single or doublestranded. Preferably, one strand of a double-stranded RNAi-inducingagent comprises at least a partial sequence complementary to a targetmRNA. The nucleotides of the inhibitory nucleic acid can be chemicallymodified, natural or artificial. The sequence homology between the RNAiinducing agent and the targeted tight junction target mRNA may be 100%or less, but is ideally greater than about 50% and typically 90% orgreater and even more preferably at least 98% and 99%. It will beunderstood that the percentage of sequence homology between RNAiinducing agent and the target mRNA should be sufficient to result insequence specific association between the RNAi inducing agent, e.g.siRNA, and the target mRNA, preferably under cytoplasmic conditions.

Such siRNAs comprise two RNA strands having a region of complementarityof approximately 20 or so nucleotides in length and optionally furthercomprises one or two single-stranded overhangs or loops. In mammaliancells, dsRNA longer than 30 base pairs can cause non-specific genesuppression by an interferon a response. However, cells transfected with21 nucleotide synthetic double-stranded siRNA bearing two nucleotidesprotruding at both 3′-ends have been found to escape an interferonresponse and effectively exert sequence-specific gene silencingfunction. The silencing effect of the synthetic siRNA, however, istransient. The double stranded siRNA molecule down regulates expressionof the tight junctions of the blood brain barrier and/or the bloodretinal barrier via RNAi, wherein each strand of said siRNA molecule isindependently about 18 to about 28 nucleotides in length and one strandof the siRNA molecule comprises a nucleotide sequence having sufficientcomplementarity to the RNA of the target tight junction protein orproteins for the siRNA molecule to direct cleavage of the target RNA viaRNA interference.

In shRNA, the single RNA strand may form a hairpin structure with a stemand loop and, optionally, one or more unpaired portions at the 5′ and/or3′ portion of the RNA.

Another post-transcriptional gene silencing process is mediated by microRNA or miRNA, an ssRNA species which suppress mRNA translation. LikesiRNA, miRNA are derived from RNA precursors that are processed to 21-25nucleotide sequences by endonuclease Dicer and form a sequence specificgene silencing complex.

Thus, the invention is directed to RNAi technology and can ideally beeffected by, for example, siRNA, miRNA and/or shRNA. For ease ofreference, siRNA and shRNA will be referred to in the followingpassages. However, it will be understood that siRNA, miRNA or shRNA orany other RNAi inducing agent may be used in the following methods.

According to a first aspect of the invention, there is provided the useof RNAi targeting tight junction proteins in a method for thereversible, transient and controlled size selective opening of theparacellular pathway of the blood brain barrier by delivery, preferablysystemic delivery, of the RNAi inducing agent, preferably siRNA orshRNA, targeting tight junction proteins to the subject.

Advantageously, the delivery of siRNA or shRNA targeting tight junctionproteins results in the controlled, reversible and transient opening ofthe paracellular pathway of the blood brain barrier to allow thepermeation of molecules, ideally less than 15 kDa, across the braincapillary endothelial or retinal endothelial cells. Ideally, thedelivery of low molecular weight drugs, such as those belowapproximately 1 kDa, is facilitated.

According to a specific embodiment of this aspect of the invention,there is provided the use of siRNA in a method for the reversible,transient and controlled size selective opening of the paracellularpathway of the blood brain barrier wherein the method comprises thedelivery, preferably systemic delivery, of siRNA targeting tightjunction proteins and results in the reversible and transientRNAi-mediated suppression of the blood brain barrier tight junctionmodulating peptide transcripts in brain capillary endothelial or retinalendothelial cells to allow the permeation of molecules, less than 15kDa, across the blood brain barrier, through brain capillary endothelialor retinal endothelial cells.

The use of RNAi inducing agents, such as siRNA or shRNA, targeting tightjunction proteins is the first time that the reversible, transient andsize-selective opening of the BBB has been achieved. This opening of theBBB has many different applications including, but not limited to thefollowing:

-   -   Use as an experimental model to study the paracellular system by        development of a conditional TJ modulating peptide knockout        mouse;    -   Use a conditional TJ modulating peptide knockout mouse as a        general experimental platform to test efficacy of a wide range        of pharmaceutical products;    -   Use to increase the permeability of the BBB to active agents        which previously would not have permeated the BBB;    -   Targeting many different TJ proteins to provide for flexibility        of molecule size that can cross the BBB; and    -   Use in the treatment of many diseases or disorders which involve        the paracellular pathway and blood brain barrier or blood        retinal barrier.

The following passages relate to RNAi technology using siRNA, however,as stated above these passages are equally applicable to RNAi technologyusing miRNA, shRNA or other RNAi inducing agents.

We have found that the opening of the paracellular pathway of the BBBoccurs approximately 24 to 48 hours after siRNA delivery. Levels ofexpression of the tight junction proteins return to normal atapproximately 72 hours post siRNA delivery and hence the BBB no longerremains open after this time period. Thus, the opening of the BBB usingRNAi is transient and reversible. This is a major advantage of thepresent invention, ensuring that the BBB integrity is restored fullypost-siRNA delivery. It is this feature of temporarily compromising theintegrity of the BBB to allow for the passage of small molecules intothe brain which provides one of the major advantages of the inventionover known techniques which opening the BBB in a potentially deleteriousnon-controlled and permanent manner

In addition, the opening of the BBB is size-selective and controlledallowing the permeation of molecules, ideally less than 15 kDa, acrossthe BBB. Ideally, molecules less that 1 kDa, preferably less than 800Da, are allowed to permeate across the BBB, through the brain capillaryendothelial or retinal endothelial cells. It will be understood that thesize-selective opening of the BBB may be different for the different TJproteins targeted. For example, Occludin allows the permeation ofmolecules of up to approximately 60 KDa to 80 KDa whereas Claudin 1, 5and 12 allow the permeation of molecules of approximately 2 KDa orlower.

Ideally, for RNAi to be effective, a large volume of siRNA isadministered to the subject to ablate/suppress the blood brainbarrier/blood retinal barrier tight junction protein transcripts inbrain capillary endothelial or retinal cells. Preferably, the amount ofsiRNA delivered is approximately 1 μg siRNA per 1 kg body weight of thesubject. However, it will be understood that other volumes of siRNA maybe contemplated. For example, for small mammals such as rodentsincluding mice, this amount may be as low as from approximately 5 μg to50 μg. Ideally, an amount of approximately 20 μg is used. For largemammals, such as humans, of typically approximately 70 kg weight, anappropriate amount of siRNA to be injected would be in the region of0.07-0.15 grams. Again, other amounts of siRNA may be contemplated.

Preferably, delivery is by systemic administration includingintra-venous delivery and intra-arterial delivery, such as intra-carotiddelivery. Administration may be direct administration or via a catheter.Ideally, the siRNA is administered to the subject by systemichydrodynamic delivery.

Hydrodynamic delivery is an efficient and inexpensive procedure whichcan be used to deliver a wide range of nucleic acids to tissues andother organs in-vivo. The successful application of hydrodynamicdelivery is dependent on the rapid injection of a large aqueous volumecontaining the oligonucleotides into the vasculature of the subject.

Essentially, systemic hydrodynamic delivery according to the presentinvention involves the intravascular administration of siRNA. In rodentssuch as mice, tail-vein delivery may be contemplated. In humans or othermammals, intra-carotid administration directly to the carotid artery orheart via the jugular vein may be contemplated. Hydrodynamic delivery inhumans could also be contemplated by administration via the hepaticportal vein following insertion of a line in the femoral vein.Specifically, delivery of siRNA to brain capillaries in humans couldpotentially be mediated via intra-carotid administration. Alternatively,the direct injection of high concentrations of siRNA in volumes, forexample up to 300-400 ml, to the heart of humans may allow for enhanceddelivery to the brain capillaries. Administration could take place byinserting a very narrow catheter into the femoral artery of the subjectand advancing it into one of the neck arteries at the base of the brain.The siRNA may then be administered through the catheter.

The efficiency of oligonucleotide (i.e., siRNA) delivery by systemichydrodynamic delivery is enhanced by increasing the volume and pressure,and hence permeability, of the tissue's blood vessel. Permeability canbe increased by the following

-   -   increasing the intravascular hydrostatic (physical) or osmotic        pressure;    -   delivering the injection fluid rapidly (injecting the injection        fluid rapidly);    -   using a large injection volume; and/or    -   increasing permeability of the tight junction following        suppression of TJ protein transcripts.

Advantageously, we have found that hydrodynamic delivery may be used forthe delivery of siRNA targeting tight junction proteins to result in thereversible and transient RNAi-mediated suppression of blood brainbarrier tight junction protein transcripts in brain capillaryendothelial or retinal cells. As explained above, this opens the BBB andallows the permeation of molecules less than 15 kDa to the braincapillary endothelial or retinal cells. Delivery to the BBB has not beenachieved before.

Ideally, for systemic hydrodynamic administration, the siRNA isdelivered in solution, preferably phosphate buffered saline solution orwater.

Ideally, the solution has a volume of between 8-10% of the body weightof the subject. We have found that this high volume delivery of siRNAdirected against selected tight junction proteins increases thepermeability of the brain microvasculature when compared tonon-targeting siRNA.

Ideally, where the subject is a large mammal, the total volume could bein the region of several litres, depending on the body weight of thesubject. Such a high volume may be desirable in situations like thetreatment of traumatic brain injury or catastrophic stroke where thereis no other treatment available and the subject would die withoutfurther intervention. Where the subject is a small mammal such as amouse, the total volume is ideally from 1 to 3 ml. The exact volume tobe delivered will depend on the body weight of the subject.

Conveniently, a specific volume is delivered within a specific timeperiod, for example a rate less than 0.05 ml per gram of mammal weightper second. The introduction of a defined volume in a short time periodaids this administration route.

According to one embodiment of this aspect of the invention, systemichydrodynamic delivery to mice involves the injection of siRNA inapproximately 1 to 3 ml of liquid into the tail vein of the mouse at arate of approximately 1 ml/second.

Alternatively, the siRNA may be administered using a non-hydrodynamicapproach involving the use of a high concentration of siRNA in a lowvolume solution. Similar concentrations of siRNA to the systemichydrodynamic approach are used. Typically, a total volume of 70 to 200ml for large mammals, such as humans, may be contemplated. Thus, thepercentage volume based on body weight used for non-hydrodynamicdelivery is much lower than hydrodynamic volumes.

An alternative route involves plasmid DNA expressing siRNA which hasbeen developed utilizing transcription systems including T7 polymerase,and mammalian pol II or pol III promoters. The effectiveness of genesilencing by siRNA-encoding plasmids depends on DNA transfectionefficiency and also results in transient siRNA expression. Suchalternative routes are expanded on later in the description.

The delivery of siRNA according to the invention results in thetransient, reversible and size-selective opening of the BBB. Thisopening may be controlled by the specific siRNA chosen and the deliveryconditions. For example, the choice of the specific TJ protein siRNA mayallow the permeation of molecules of different sizes to the brainendothelial or retinal endothelial cells of the BBB.

Furthermore, this invention represents a non-invasive technique for thedelivery of small molecules to all areas of the brain or retina wherethey would otherwise be excluded. Previously, this has not beenpossible, and known methods for the delivery of agents to the BBB areinvasive and carry many risks. For example, temporarily shrinking theBBB cells with a concentrated sugar solution has been used to disruptthe BBB in the treatment of brain tumours. This temporarily allowschemotherapy drugs to pass into the brain and reach the tumour. However,such a treatment carries significant risks and can only be used incertain circumstances. Furthermore, the BBB is disrupted in a non-sizeselective manner and this method only allows for a small time framewithin which to deliver a drug of interest(www.ohsu.edu/bbb/bbbdtherapy.html). Thus, the provision of alternativetherapies to open the BBB which can be controlled and do not have suchsevere side-effect for opening the blood brain barrier is highlydesirable.

In addition, known TJ-associated protein knockout mice have majordisadvantages in terms of compromised and deleterious BBB functionalityand mortality associated with these knockout mice is high. The presentinvention overcomes these problems by providing for a transient knockoutthereby overcoming the mortality associated with known TJ proteinknockout mice.

The RNAi technique of the invention, whether siRNA, miRNA or shRNA orother RNAi inducing agent, targets TJ associated proteins from the bloodbrain barrier and/or blood retinal barrier. For example, the siRNA usedto open the BBB is targeted to a TJ protein. Ideally, the tight junctionproteins are selected from transmembrane proteins associated with thetight junction of the brain microvasculature. Typically the region ofthe siRNA sequence with sequence identity to the target mRNA, the tightjunction protein transcripts, is from 14 to 30 nucleotides in length,for example from 16 to 24 nucleotides, more preferably from 18 to 22nucleotides, most preferably from 19 to 21 nucleotides in length. ThesiRNA is sufficiently complementary to the target mRNA of the tightjunction protein that the siRNA agent silences production of a proteinencoded by the target mRNA. The siRNA may be blunt ended or may haveoverhangs at its 3′ or 5′ termini, preferably at both of its termini.The overhangs are preferably short in length, for example less than 30nucleotides, preferably less than 20 nucleotides more preferably lessthan 10 nucleotides, even more preferably less than 5 nucleotides, mostpreferably less than 3 nucleotides in length. Typically, the overhangsare two nucleotides in length.

Thus, the siRNAs of the invention are typically less than 30 nucleotidesin length and can be single or double stranded. Longer siRNAs cancomprise cleavage sites that can be enzymatically or chemically cleavedto produce siRNAs having lengths less than 30 nucleotides, typically 21to 23 nucleotides as above. It will be understood that siRNAs sharesequence homology with corresponding target mRNAs. The sequence homologycan be 100% or less and should be sufficient to result is sequencespecific association between the siRNA and the targeted mRNA. ExemplarysiRNAs do not activate the interferon signal transduction pathway. Themost preferred embodiment of the invention comprises a siRNA having 100%sequence identity with the target mRNA, the tight junction protein.However, other sequences with less than 100% homology (as described inrelation to RNAi inducing agents in general) may be used wherein thesiRNA is of sufficient homology to guide the RNA-induced silencingcomplex (RISC) to the target mRNA for degradation.

Limited mutations in siRNA relative to the target mRNA may also becontemplated. It will be understood that the siRNA of the presentinvention ideally has nucleotide overhangs. For example, the siRNA mayhave two nucleotide overhangs (e.g. UU), thus, the siRNA will comprise a21 nucleotide sense strand and a 21 nucleotide antisense strand pairedso as to have a 19 nucleotide duplex region. The number of nucleotidesin the overhang can be in the range of about 1 to about 6 homologousnucleotide overhangs at each of the 5′ and 3′ ends, preferably, about 2to 4, more preferably, about 3 homologous nucleotide overhangs at eachof the 5′ and 3′ ends.

In addition, the siRNA may be chemically modified, for example, to bemore stable upon administration The nucleotides overhang can bemodified, for example to increase nuclease resistance. For example, the3′ overhang can comprise 2′ deoxynucleotides, e.g., TT, for improvednuclease resistance.

One of these transmembrane proteins includes junctional adhesionmolecule (JAM). Alternatively, the tight junction-associated moleculesare selected from one or more of the following occludins, claudinsand/or zonula-occludens (ZO-1, ZO-2, ZO-3).

Sequences of exemplary siRNAs and the associated target sequence areprovided below.

In the alignments depicted below, the sense sequence and the antisensesequence are denoted in the Application in the 5′-3′ direction, but in adouble-stranded siRNA molecule, the sense and anti-sense sequences areoriented in opposite directions to one another, i.e., the sense strandis oriented 5′-3′ and the anti-sense strand is oriented 3′-5′ (relativeto the sense strand). Thus, in the first example, the “sense” sequence(SEQ ID NO:9) is transcribed from the “target” sequence (SEQ ID NO: 32)in the opposite direction to the “antisense” sequence (SEQ ID NO:10).The sense and anti-sense sequences, when separated by a hairpin loop,hybridise to form siRNA molecules.

According to one specific embodiment of this invention, the tightjunction associated molecule is chosen from one or more of claudin 1 to19 and/or 21. Preferably, the tight junction associated molecule isclaudin 1, 5 and/or 12.

According to a preferred embodiment of the invention the tight junctionassociated molecule is claudin-5. Ideally, the siRNA is selected fromconserved regions of the Claudin-5 gene.

Specifically, the claudin-5 siRNAs may have the following sequence (5′to 3′):

(SEQ ID No. 1) Sense sequence: CGUUGGAAAUUCUGGGUCUUU (SEQ ID No. 2)Antisense sequence: AGACCCAGAAUUUCCAACGUU (SEQ ID No. 3) Sense sequence:CAAUGGCGAUUACGACAAGUU (SEQ ID No. 4) Antisense sequence:CUUGUCGUAAUCGCCAUUGUU (SEQ ID No. 5) Sense sequence:UCACGGGAGGAGCGCUUUAUU (SEQ ID No. 6) Antisense sequence:UAAAGCGCUCCUCCCGUGAUU (SEQ ID No. 7) Sense sequence:GCGCAGACGACUUGGAAGGUU (SEQ ID No. 8) Antisense sequence:CCUUCCAAGUCGUCUGCGCUU

According to a still preferred embodiment of the invention the tightjunction associated molecule is claudin-1. Ideally, the siRNA isselected from conserved regions of the Claudin-1 gene. Specifically, theclaudin-1 siRNA has the following sequence (5′ to 3′):

CLDN1 (1) target sequence: GCAAAGCACCGGGCAGAUA (SEQ ID NO. 32):(SEQ ID No. 9) Sense sequence: AUAGACGGGCCACGAAACGUU (SEQ ID No. 10)Anti-sense strand: CGUUUCGUGGCCCGUCUAUUUCLDN1 (2) target sequence: GAACAGUACUUUGCAGGCA (SEQ ID NO. 33):(SEQ ID No. 11) Sense strand: ACGGACGUUUCAUGACAAGUU (SEQ ID No. 12)Anti-sense strand: CUUGUCAUGAAACGUCCGUUUCLDN1 (4) target sequence: UUUCAGGUCUGGCGACAUU  (SEQ ID NO. 34):(SEQ ID No. 13) Sense sequence: UUACAGCGGUCUGGACUUUUU (SEQ ID No. 14)Anti-sense strand: AAAGUCCAGACCGCUGUAAUU

According to a still preferred embodiment of the invention the tightjunction associated molecule is Occludin. Ideally, the siRNA is selectedfrom conserved regions of the Occludin gene. Specifically, the OccludinsiRNA has the following sequence (5′ to 3′):

Occl (1) target sequence: GUUAUAAGAUCUGGAAUGU (SEQ ID NO. 35):(SEQ ID No. 15) Sense sequence: UGUAAGGUCUAGAAUAUUGUU (SEQ ID No. 16)Anti-sense sequence: CAAUAUUCUAGACCUUACAUUOccl (2) target sequence: GAUAUUACUUGAUCGUGAU (SEQ ID NO. 36):(SEQ ID No. 17) Sense sequence: UAGUGCUAGUUCAUUAUAGUU (SEQ ID No. 18)Anti-sense sequence: CUAUAAUGAACUAGCACUAUUOccl (3) target sequence: CAAAUUAUCGCACAUCAAG (SEQ ID NO. 37):(SEQ ID No. 19) Sense sequence: GAACUACACGCUAUUAAACUU (SEQ ID No. 20)Anti-sense sequence: GUUUAAUAGCGUGUAGUUCUUOccl (4) target sequence: AGAUGGAUCGGUAUGAUAA (SEQ ID NO. 38):(SEQ ID No. 21) Sense sequence: AAUAGUAUGGCUAGGUAGAUU (SEQ ID No. 22)Anti-sense sequence: UCUACCUAGCCAUACUAUUUU

According to a preferred embodiment of the invention the tight junctionassociated molecule is claudin-12. Ideally, the siRNA is selected fromconserved regions of the Claudin-12 gene.

Specifically, the claudin-5 siRNAs may have the following sequence (5′to 3′):

CLDN12 SIRNA (1) Target sequence: (SEQ ID NO. 39) GAAAUCGACAUUCCAGUAG(SEQ ID No. 24) 5′-GAAAUCGACAUUCCAGUAGUU-3′ (SEQ ID No. 25)5′-CUACUGGAAUGUCGAUUUCUU-3′ CLDN12 SIRNA (2) Target sequence:(SEQ ID NO. 40) CGUGGUACCUGUCGGUUGA (SEQ ID No. 26)5′-CGUGGUACCUGUCGGUUGAUU-3′ (SEQ ID No. 27) 5′-UCAACCGACAGGUACCACGUU-3′CLDN12 SIRNA (3) Target sequence: (SEQ ID NO. 41) GUAACACGGCCUUCAAUUC(SEQ ID No. 28) 5′-GUAACACGGCCUUCAAUUCUU-3′ (SEQ ID No. 29)5′-GAAUUGAAGGCCGUGUUACUU-3′ CLDN12 SIRNA (4) Target sequence:(SEQ ID NO. 42) GGUCUUUACCUUUGACUAU (SEQ ID No. 30)5′-AAUCUUUACCUUUGACUAUUU-3′ (SEQ ID No. 31) 5′-AUAGUCAAAGGUAAAGAUUUU-3′

The techniques of designing siRNA are well known to those skilled in theart and will not be expanded on in detail here.

It will be understood that the siRNA used in the present invention maytarget a single TJ modulating peptide. Alternatively, one or more siRNAstargeting different TJ proteins may be used concurrently. For example,siRNA targeting several different types of Claudin proteins may becontemplated. When using combinations siRNA targeting different TJproteins, the crucial aspect is that the integrity of the overall tightjunction should be preserved.

According to one embodiment of this aspect of the invention,combinations of siRNA could include claudin-1 with claudin-5, claudin-12with claudin-5, claudin-12 with claudin-1. Alternatively, claudin-1,claudin-5 and claudin-12 may be used together. Occludin may also becombined with one or more Claudin types. It is envisaged that thesecombinations further increase permeability at the BBB in a controlledand size-selective nature.

shRNA may also be chosen to target these TJ proteins. shRNA targeting TJproteins will ultimately have the same sense and anti-sense sequence asthe siRNA. The only difference is that they contain short hairpinscomposed of the following nucleotides UAUCAAGAG which form a hairpinstructure and allow for them to be cloned into delivery vectors.

Ideally, an inducible vector is used for shRNA delivery to prevent theotherwise continuous expression of the shRNA targeting a TJ protein ofinterest and resultant continuous suppression of these TJ targetingproteins. For example, AAV-mediated delivery which will be highlylocalised to specific regions of the brain or retina may be used todeliver shRNA. Preferably, inducible AAV vectors which allow for theinduced expression of shRNA targeting TJ proteins when specific drugsare administered are used. Such inducible AAV vectors enable thetransient suppression of the tight junction targeting proteins, such asclaudin-5.

According to a second aspect of the present invention, the delivery ofthe RNA inducing agent, such as siRNA, miRNA or shRNA etc, according tothe invention may be used in the generation of an experimental animalmodel used for studying the action of the paracellular pathway and thephysiology of the BBB. This type of animal model overcomes highmortality rates associated with the known BBB knockout mouse as itreversibly, transiently and in a controlled size selective manner opensthe paracellular pathway of the BBB. Such a conditional tight junctionprotein knockout mouse which transiently suppresses the BBB tightjunction proteins to open the BBB can be used to test the efficacy of awide range of pharmaceutical products (including such products whichpreviously could not permeate the BBB) and/or study the paracellularsystem.

As such, this method can be used in an animal model for the testing ofvarious active agents which have previously not been able to penetratethe BBB and the generation of new treatments of diseases and disorderswhich affect brain and retinal function. Advantageously, this methodenables the generation of an ideal experimental platform for theassessment of a wide range of pharmacological agents which wouldotherwise not traverse the blood-brain barrier. Thus, this method couldallow for the establishment of experimental animal models, forneurodegenerative and neuropsychiatric disorders etc.

According to a third aspect of this invention, there is provided the useof an RNAi inducing agent, such as siRNA, miRNA or shRNA etc, targetingtight junction proteins in the manufacture of a medicament for thetreatment of disease or disorder of the brain or retina wherein themethod comprises the reversible, transient and controlled size selectiveopening of the paracellular pathway of the blood brain barrier by thedelivery, preferably systemic delivery, of the siRNA targeting tightjunction proteins to result in the reversible and transientRNAi-mediated suppression of blood brain barrier tight junction proteintranscripts in brain capillary endothelial cells and/or retinalendothelial cells and allow the permeation and delivery of an activeagent, less than 15 kDa, directed to the treatment of the disease ordisorder of the brain or retina.

The RNAi inducing agent, preferably siRNA (miRNA or shRNA etc),targeting the tight junction proteins transiently opens the blood brainbarrier to allow delivery of the active agent across the blood brainbarrier. Ideally, the method comprises the sequential administration ofthe active agent after administration of the siRNA or shRNA. Thisensures that paracellular pathway is open when the active agent isadministered and the active agent can permeate through the braincapillary endothelial cells and/or retinal endothelial cells to reachthe brain and/or retina. However, it may also be contemplated that thedelivery of the active agent takes place before orconcurrently/simultaneously with the RNAi inducing agent.

This aspect of the present invention is applicable for the treatment ofmany diseases or disorders where the BBB or BRB is implicated. Theseinclude but are not limited to neurodegenerative disorders (such asAlzheimer's disease, multiple sclerosis etc), stroke and traumatic braininjury (TBI), and infectious processes and inflammatory pain, retinaldisorders including age-related macular degeneration (AMD), glaucoma anddiabetic retinopathy. For example, the present invention may be used forthe controlled delivery of therapeutic agents to the central nervoussystem (CNS) in a range of neurodegenerative or other conditions thatcurrently offer little or no prospect of effective treatment.

According to this aspect of the present invention, the treatment of theparticular disease generally involves the opening of the BBB with orfollowed by delivery of an active agent across the BBB. The active agentis ideally delivered after the opening of the BBB, i.e. after 24 or 48hours post-siRNA delivery. Alternatively, as mentioned before the activeagent many be co-administered with or prior to the RNAi inducing agent.

The active agent may be chosen from conventional pharmaceuticals, suchas agents that modulate neuronal function, chemotherapeutic agents,anti-tumour agents, agents that modulate retinal function andnon-steroidal anti-inflammatories (NSAIDs). As such, the active agentmay be any conventional biologically active therapeutic agent.

Alternatively, the active agent may be a hypertonic solution, preferablya hypertonic saline or sugar solution. Such hypertonic solutions may beused in the treatment of traumatic brain injury to allow for water to bedriven out of the brain following injury, and may significantly preventthe occurrence of cerebral oedema. Mannitol, a sugar solution, may alsobe used in the present invention. Mannitol is used in conventional“mannitol osmotherapy” however, mannitol when used conventionally doesnot cross the BBB. Mannitol acts as an osmotic diuretic agent and a weakrenal vasodilator.

It will be understood that the active agent may also be a smallmolecule, antisense oligonucleotide, ribozyme or protein, polypeptide orpeptide.

According to a preferred embodiment, the active agent is a further RNAiinducing agent, including siRNA, miRNA or shRNA etc, which targets thedisease or disorder being treated, such that after opening of the BBB,further RNAi inducing agents could be delivered into the brain to treatthe particular disease or disorder.

This aspect of the present invention will now be discussed in relationto several specific diseases or conditions and one RNAi inducing agentsiRNA, although other RNAi inducing agents such as shRNA or miRNA couldalso be used.

The stress response in traumatic brain injury (TBI) is manifested by thecessation of water diffusion across the BBB, leading to acute increasesin intracranial pressure and cerebral oedema. Conventional therapies forthe management of cerebral oedema and raised intracranial pressurefollowing TBI involve hyperosmolar and hypertonic therapy, includingsaline or sugar therapy. For example, the osmotic diuretic mannitol iscommonly used to treat TBI as it establishes an osmotic gradient betweenplasma and brain cells and draws water across the BBB into the vascularcompartment. Alternatively, hypertonic saline produces a reduction incerebral oedema by moving water out of cells, reducing tissue pressureand cell size. For the acute treatment of TBI, patients are givenmannitol and hypertonic saline to try and resolve the osmotic shift inwater diffusion in the brain.

However, mannitol and hypertonic saline will only be effective for up to24 hours and if swelling occurs for longer than this period, patientswill either die or be left with permanent brain damage. Thus, bothmannitol and hypertonic saline have significant disadvantages in termsof side-effects, such as severe intravascular volume depletion,hypotension and hyperkalemia, and there is difficulty in ascertainingthe correct dosage needed.

Furthermore, temporarily shrinking the BBB cells with a concentratedsugar solution has been used to disrupt the BBB in the treatment ofbrain tumours as expanded on before can have significant deleteriousside effects, is only used as a last resort and is not suitable for allpatients. Indeed the use of mannitol to open the BBB does not result inselective controlled opening of the BBB and can result in further damageto the brain. Thus, there is a need to provide an alternative therapyfor dealing with TBI or stroke.

According to this aspect of the present invention, there is provided theuse of siRNA in the manufacture of a medicament for the treatment of atraumatic brain injury or stroke wherein the method comprises thereversible, transient and controlled size selective opening of theparacellular pathway of the blood brain barrier by the delivery,preferably systemic delivery, of the siRNA targeting tight junctionproteins to result in the reversible and transient RNAi-mediatedsuppression of blood brain barrier tight junction protein transcripts inbrain capillary endothelial cells and optionally following theadministration of an active agent, such as a hypertonic sugar or salinesolution, to allow the permeation and free diffusion of water across theblood brain barrier, reduction of intracranial pressure and/or reductionof cerebral oedema.

This ability to allow the transient, controlled and reversible freepermeation and free diffusion of water across the blood brain barrier isvery important in conditions such as traumatic brain injury (TBI) orcatastrophic stroke. In these conditions, the cessation of waterdiffusion from the brain to the blood can cause increase intracranialpressure, leading to cerebral oedema and possibly death or severedisability. The present invention provides a new treatment to allowbrain-to-blood diffusion of water and reduce the effects of intracranialpressure and/or cerebral oedema. Thus, advantageously and unexpectedly,the use of siRNA targeting tight junction proteins enhances waterdiffusion across the BBB. This provides an alternative means forintervention in cerebral oedema associated with TBI, acute TBI inparticular. The use of an RNAi inducing agent targeting tight junctionproteins allows the free diffusion of water from the brain of subjectswith TBI in a controlled and reversible manner for a period of up to 72hours when the BBB is open. An active agent, such as a hypertonicsolution (e.g. mannitol or hypertonic saline) may optionally be usedtogether with the siRNA to provide an osmotic gradient to facilitatewater diffusion initially until the BBB opens after 24/48 hours.

Thus, it will be understood that the siRNA targeting the tight junctionproteins may be used on its own or in combination (either sequentiallyor simultaneously) with conventional TBI or stroke therapies, such ashyperosmolar and hypertonic saline/sugar therapy or mannitolosmotherapy.

According to a specific embodiment, the method of the invention allowsfor water flux from the brain to the blood in a highly controlled mannerand may be used in combination with mannitol/hypertonic sugar or salinetherapy. Advantageously, the siRNA and mannitol/hypertonic salinetherapy may be infused at the same time, and as the mannitol/hypertonicsaline stops working after approximately 24 hours, the controlledopening of the BBB commences to further allow water flux from the brainthrough the BBB.

According to another aspect of the present invention, there is providedthe use of siRNA in the manufacture of a medicament for the treatment ofa neurodegenerative or neuropsychiatric disorder wherein the methodcomprises the reversible, transient and controlled size selectiveopening of the paracellular pathway of the blood brain barrier by thedelivery, preferably systemic delivery, of siRNA targeting tightjunction proteins to result in the reversible and transient RNAimediated suppression of the blood brain barrier tight junction proteintranscripts in brain capillary endothelial cells and allows thepermeation and delivery of an active agent less than 15 kDa whichmodulates neuronal function to the brain capillary endothelial cells.

The agent which modulates neuronal function may be any conventionaltreatment for conventional neurodegenerative or neuropsychiatricdisorders.

Age-related macular degeneration (AMD) affects more than 1.75 millionindividuals in the United States and is the leading cause of visionimpairment and blindness in persons 60 years or older. The greatestknown risk factor for developing AMD is advanced age, however, ocularrisk factors for exudative AMD include the presence of soft drusen,macular pigment changes, and choroidal neovascularization. Additionalrisk factors associated with AMD include smoking, obesity, hypertensionand positive family history. AMD presents in two basic forms: dry or wetAMD, the latter being associated with vascular permeability andhemorrhages. In the more severe, exudative form, new vessels originatingfrom the choriocapillaris bed develop under the macula of the retina,growing into the sub-retinal space between the retina and the retinalpigmented epithelium (RPE). These newly sprouted vessels leak serousfluid and blood under the neurosensory retina and lead to macular edemaand retinal detachment causing symptoms of visual distortion(metamorphosia) and blurring of vision.

Glaucoma is a complex disease, which may involve degeneration of thetrabecular meshwork and/or lamina cribrosa of the eye, resulting inaberrant function of drainage channels and/or degeneration of the opticnerve head. As a result, ganglion cells (the output neurons of theretina) die, resulting in narrowing of and/or loss of the visual fields,leading, if untreated, to severe visual handicap in a significantproportion of cases.

The majority of cases of open-angle glaucoma involve increasedintraocular pressure although a growing number of so-called normalpressure glaucomas are now being identified. In those cases where apressure build up is registered, pressure-reducing eye drops are oftenof substantial value in slowing down the progression of the disease.

However, surgical intervention is sometimes required to alleviateintraocular pressure and some forms of open angle glaucoma becomerefractory to treatment.

Open angle glaucoma affects up to 1 million persons within the BritishIsles at the present time. While most forms of the disease aremultigenic or multifactorial, some forms of the diseases are inheritedaccording to apparent mendelian ratios, i.e., they are transmitted in anautosomal dominant sense. In some such forms of disease, mutationswithin the so-called myocilin gene have been identified (Stone et al,Science, 275, 1997, 668-670). Moreover, in up to 4% of multifactorialforms of disease, similar mutations have been encountered. Thus, 40,000persons, or more, within the British Isles, have a form of glaucomacaused my mutations within the myocilin gene.

Diabetic retinopathy, eye damage that frequently occurs as a result ofdiabetes, is related to the breakdown of the blood-retinal barrier. Thebarrier becomes more leaky in patients with diabetic retinopathy.

According to this aspect of the present invention, there is provided theuse of RNAi in the manufacture of a medicament for the treatment of adisease of the retina wherein the method comprises the reversible,transient and controlled size selective opening of the paracellularpathway of the blood brain or blood retinal barrier wherein the methodcomprises the delivery, preferably systemic delivery, of siRNA targetingtight junction proteins which results in the reversible and transientRNAi mediated suppression of the blood brain or blood retinal barriertight junction protein transcripts in brain capillary endothelial cellsand/or retinal endothelial cells and allow the permeation and deliveryof active agent which modulates retinal function, less than 15 kDa,across the brain capillary endothelial cells and/or retinal capillaryendothelial cells.

This aspect of the invention, involves the treatment of a disease of theretina by opening the BBB or BRB to allow the permeation and delivery ofactive agent which modulates retinal function across the retinalcapillary endothelial cells.

The agent which modulates retinal function may be any conventionaltreatment for the above conditions. Additionally, for example, vascularendothelial growth factor receptor (VEGF) dysregulation is a keymediator of age-related macular degeneration (AMD), thus, increaseddelivery of these inhibitors to the retina may significantly retard theprogression of AMD. Until now, these small molecule inhibitors of theVEGF receptor were not able to cross the blood retinal barrier. This isa major advantage of the present invention.

According to yet another embodiment of the present invention, there isprovided the use of siRNA in the manufacture of a medicament for thetreatment of a brain tumor wherein the method comprises the reversible,transient and controlled size selective opening of the paracellularpathway of the blood brain barrier by the delivery, preferably systemicdelivery, of siRNA targeting tight junction proteins which results inthe reversible and transient RNAi-mediated suppression of the bloodbrain barrier tight junction protein transcripts in brain capillaryendothelial cells and allows the permeation and delivery of ananti-tumor or chemotherapeutic agent less than 15 kDa to the braincapillary endothelial cells.

Thus, this aspect of the present invention provides for the transientopening of the BBB/BRB which can be used for the enhanced delivery ofconventional chemotherapeutic or anti-tumour drugs which would normallybe redundant for the treatment of conditions such as brain tumours asuntil now they have been unable to cross the BBB.

In a typical embodiment of all aspects of the invention, the subject isa mammal such as a cow, horse, mouse, rat, dog, pig, goat, or a primate(Macaque). In a much preferred embodiment, the subject is a human, e.g.a normal human or human diagnosed with or predicted to have a disease ordisorder that is currently un-treatable due to the non-availability ofdrugs that cross the BBB.

According to a fourth aspect of the invention, there is provided apharmaceutical composition comprising a pharmaceutically acceptablesolution of siRNA targeting tight junction proteins to result in thereversible, transient and controlled size selective opening of theparacellular pathway of the blood brain barrier suitable for deliveryand an active agent for the treatment of a defined disease or disorder.

The active agent may be chosen from conventional pharmaceuticals, suchas active agents that modulate neuronal function, chemotherapeuticagents, anti-tumour agents, agents that modulate retinal function andnon-steroidal anti-inflammatories (NSAIDs) or hypertonic solution asdefined previously. Specific examples are given in the above passages.

Alternatively or additionally, the active agent may also be a smallmolecule, antisense oligonucleotide, ribozyme or protein, polypeptide orpeptide. Ideally, the active agent is a further siRNA which targets thedisease or disorder being treated, such that after opening of the BBB,further siRNA molecules could be delivered into the brain to treat theparticular disease or disorder.

Ideally, the pharmaceutical composition is adapted for systemichydrodynamic delivery and is present in a pharmaceutically acceptablecarrier.

It will be understood that the siRNA and active agent may be suitablefor simultaneous or sequential administration. Thus, the siRNA may beadministered alone or in combination with an active agent. Although, thesequential administration after the BBB has opened is the preferreddelivery method for some active agents.

According to yet another aspect of the present invention, there isprovided a method for the reversible, transient and controlled RNAimediated size selective opening of the paracellular pathway of the bloodbrain barrier comprising the steps of the delivery, preferably systemicdelivery, of an effective amount of siRNA targeting tight junctionproteins to result in the transient and reversible RNAi mediatedsuppression of blood brain barrier tight junction protein transcripts inbrain capillary endothelial or retinal cells and to allow the permeationof molecules less than 15 kDa to brain capillary endothelial or retinalcells.

According to a further aspect of the present invention, there isprovided a method for the treatment of a disease or disorder comprisingthe reversible, transient and controlled RNAi-mediated size selectiveopening of the paracellular pathway of the blood brain barrier whereinthe method comprises identifying a subject at risk for developing thedisease or disorder;

administering an effective amount of an RNAi inducing agent, preferablysiRNA, miRNA or shRNA etc, targeting tight junction proteins bydelivery, preferably systemic delivery, of the RNAi inducing agent toresult in the transient and reversible RNAi-mediated suppression ofblood brain barrier tight junction protein transcripts in braincapillary endothelial or retinal endothelial cells and allow thepermeation of active agents used in the treatment of the disease ordisorder less than 15 kDa to the brain capillary endothelial and/orretinal cells; and administering an active agent suitable for thetreatment of the disease or disorder.

Advantageously, this method increases the permeability of the BBB todrugs or other active agents.

We have previously covered both hydrodynamic and non-hydrodynamicdelivery of siRNA to the region of interest, i.e. the BBB.

Other delivery methods could be contemplated, such as transcellular,receptor-mediated, delivery of molecules across the BBB. For example,siRNA may be delivered using electroporation or lipid mediatedtransfection. Additional delivery methods include the use of cationicpolymers, modified cationic polymers, peptide molecular transporters,lipids, liposomes, non-cationic polymers and/or viral vectors fordelivery of the RNAi inducing agent.

Further delivery methods include encapsulating or conjugating the siRNAso that delivery to the BBB is affected. As previously described above,genetically engineered proteins termed

“Molecular Trojan horses” could be used to affect delivery to the BBB.There are now numerous methods whereby siRNAs can be chemically modifiedwith, for example, cholesterol moieties in order to allow for theirdiffusion across the plasma membrane of cells. In principle, thesecholesterol conjugated siRNAs targeting claudin-5 or other tightjunction proteins could be administered without the need for ahydrodynamic injection.

Other viral mediated delivery systems may be contemplated. For example,targeted delivery of proteins across the BBB could be affected by alentivirus vector system.

Alternatively, mosaic vector particles have previously been described,and show significant promise for targeted delivery of adeno-associatedvirus (AAV) particles specifically to the vasculature (Stachler M D etal., 2006). Moreover, Work L M et al (2006) have shown that generatingdistinct capsid modifications on AAV particles will allow for targetingof viral vectors to specific viral beds including those associated withthe brain. This technique is required when shRNA is used where the shorthairpin is used for cloning into the delivery vectors. As discussedabove such an inducible vector system provides for the controlled andtransient suppression of the blood brain barrier.

Thus, according to this aspect of the invention, endothelial cellspecific AAV could be generated containing antibiotic/drug-inducibleshRNA (short hairpin RNA) sequences specific for the suppression ofclaudin-5 to provide a method for the inducible opening of the BBB orBRB following infection of brain microvascular endothelial cells withAAV containing shRNA against claudin-5. Other viral vectors may also becontemplated.

Furthermore, recently Szymanski et al., (2007) reported the developmentof an inducible plasmid pBRES which is controlled by the FDA-approveddrug mifepristone (MFP) and, in principle, is small enough to bepackaged into and subsequently delivered by AAV viral particles. pBRESis modular in design, consisting of a transactivator-inducer protein,TA, driven by a promoter of choice which interacts with the drug MFP toallow expression of the transgene of interest, i.e. shRNA, (from aregulated promoter (e.g. 6×Gal4/TAT). As this minimal promoter has beendesigned using standard Pol II elements it is unlikely to driveconventional shRNA genes which require Pol III promoters, the shRNAsequences should be cloned with micro RNA (miRNA) backbones (shRNAmirs)into pcDNA 6.2-GW/EmGFP-miR (BLOCK-iT Pol II miRNAi Expression VectorKit; Invitrogen cat. no. K4935-00). The resulting EmGFP-tagged shRNAmircan then be excised and inserted into pBRES after the regulatedpromoter. In addition, in order to add a further safeguard againstinappropriate expression of shRNA, the TA protein (which in turn willlead to shRNA expression induction) is driven using the promoter fortransferrin, claudin-12, Tie-2 or p-glycoprotein which drive highexpression in brain endothelium when compared to other tissues. Putativepromoters from these genes derived following bioinformatic analyses canthen be tested for efficiency and tissue specificity at drivingexpression of EGFP in HUVEC (ATCC no. CRL-1730) and bend. 3 (ATCC no.CRL-2299) endothelial cell lines and the most efficient promoter will beincorporated into the inducible system to drive the TA protein. Finally,the resulting MFP-inducible, endothelial-specific shRNA-expressing pBRESplasmid can be linearised and cloned into the AAV shuttle vector pAAV2CMV and packaged into the capsid modified AAV1 for delivery to thevascular endothelium.

To date, a major limitation however in the use of AAV-1 has been thelack of an efficient small-scale purification strategy. Recentlyhowever, it has been disclosed that two AAV-1 capsid proteinmodifications can enhance vascular gene transfer significantly whilealso allowing easy purification of vector particles. Using mosaic vectorparticles comprised of capsid proteins containing the well-characterizedRGD4C modification to target integrins present on the luminal surface ofendothelial cells lining the vasculature, and capsid proteins containinga modification that allows for metabolic biotinylation and efficientpurification of mosaic particles by avidin affinity chromatography, itis now possible to generate modified AAV-1 particles which willspecifically transduce endothelial cells with a high efficiency(Stachler & Bartlett, 2006). Thus, the use of AAV-1 for delivery ofshRNA in particular to the BBB and/or BRB is now feasible in practice.

In the specification, the terms “comprise, comprises, comprised andcomprising” and any variation thereof and the terms “include, includes,included and including” and any variation thereof are considered to betotally interchangeable and they should all be afforded the widestinterpretation.

The invention is not limited to the embodiments described above but maybe varied within the scope of the claims.

REFERENCES

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The present invention will now be described with reference to thefollowing non-limiting figures and examples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show the results of the quantification of claudin-5protein and mRNA levels.

FIG. 1A is a western blot analysis of claudin-5 expression 24, 48 and 72hours post hydrodynamic tail vein delivery of siRNA. Controls usedincluded an un-injected control, PBS injected control and non-targeting(Rhodopsin) siRNA injected control mice. Western blot analysis ofclaudin-5 expression 24 hours post delivery of siRNA, showed a decreasein expression when compared to un-injected, PBS injected andnon-targeting siRNA injected mice. This suppression was also evident 48post injection (CLDN5 A+B; lysates from 2 different mice). Levels ofclaudin-5 were similar to the control groups 72 hours and 1 week postdelivery of caludin-5 siRNA when compared to the corresponding levels ofβ-actin in the same lane (FIG. 1 A).

FIG. 1B shows RT-PCR analysis of claudin-5 mRNA post-injection of siRNAcompared to the control groups—un-injected control, PBS injected controland non-targeting (Rhodopsin) siRNA injected control mice. RT-PCRanalysis showed levels of claudin-5 mRNA to be significantly decreased24 hours post-injection of siRNA compared to the control groups withP=0.0427 (*) following ANOVA with a Tukey-Kramer post-test, while alsoshowing suppression at 48 hours post-injection of claudin-5 siRNA withP=0.0478 (*). Levels of claudin-5 mRNA, 72 hours (P=0.0627) and 1 week(P=0.2264) post injection were not significantly changed compared to thenon-targeting control group, showing P values greater than 0.05,representing insignificance (FIG. 1 B).

FIG. 2 shows the results of immunohistochemical analysis of claudin-5expression and localisation in the microvessels of the brain revealed acontinuous and distinct pattern of staining in the microvasculature ofthe brain in the un-injected, PBS injected and non-targeting controlmice at all time points (Red=Claudin-5; Blue-DAPI=nuclei). This patternof staining appeared decreased and non-continuous 24 hours post deliveryof claudin-5 siRNA, with a striking decrease in expression 48 hoursafter injection. The appearance of claudin-5 staining 72 hourspost-injection of claudin-5 siRNA was evident, yet non-continuous,however, 1 week post-injection, claudin-5 expression appeared similar tothat of the control groups. Scale bar approx. 20 μm. These results arerepresentative of at least 5 separate experiments.

FIGS. 3A and 3B show the results of Claudin-1, Tie-2 and Occludinexpression following suppression of claudin-5. Western blot analysis ofclaudin-1 (23 kDa) expression 24, 48, 72 hours and 1 week post deliveryof claudin-5 siRNA, showed no changes at any time points. When blotswere probed with an anti-Tie-2 (140 kDa) antibody, no distinct changesin the levels of expression of this endothelial cell specific tyrosinekinase receptor were observable at any time point or with any treatment(FIG. 3A). Levels of expression of the tight junction protein occludin(approximately 60 kDA) were also shown to remain un-changed at all timepoints post-delivery of siRNA (FIG. 3B).

FIGS. 4A, 4B, 4C and 4D show the results of Claudin-1 and Claudin-5double immunostaining in brain cryosections. Following injection ofsiRNA targeting claudin-5, and using the appropriate controls, braincryosections were stained with a rat anti-claudin-1 antibody and arabbit anti-claudin-5 antibody. Secondary antibodies used were rat IgG(Cy3; Red) and rabbit IgG (Cy2; Green). Similar to findings in FIG. 2,the pattern of claudin-5 staining appeared highly fragmented anddiscontinuous 48 hours (FIG. 4B) after injection of siRNA. Theappearance of claudin-5 staining 72 hours post-injection of claudin-5siRNA was evident, yet not as intense as the control groups (FIG. 4C).At each time point post-injection, levels of expression of claudin-1appeared to remain similar to those observed in the control groups.

FIG. 5 shows the results of an assessment of BBB integrity to a moleculeof 443 Daltons. BBB integrity was observed as green fluorescence withinthe microvessels in all control groups. However, 24 hours post-injectionof siRNA targeting claudin-5, fluorescence detected was diffuse andoutside of the microvessels in contrast to the control groups at thesame time point. At 48 hours post-injection of claudin-5 siRNA, thedistribution of the biotinylated molecule was abundant in the brainparenchyma, while this permeability was still evident 72 hourspost-delivery of siRNA when compared to the control groups. In mice 1week post-injection of siRNA targeting claudin-5, it was observed thatthe biotinylated reagent did not deposit in the parenchyma followingperfusion for 5 minutes. The EZ-Link TM Sulfo-NHS-Biotin was observedwithin the microvessels of the brain. Scale bar for 24 and 48 hour timepoints approx 200 μm. Scale bar for 72 hour and 1 week time pointsapprox 100 μm. All tracer experiments were repeated in mice at least 5times.

FIGS. 6A and 6B show the results of extravasation of Hoechst H33342 dyefrom brain and retinal microvessels, by showing Hoechst 33342 and FD-4co-perfusion 24, 48, 72 and 1 week post-hydrodynamic tail delivery ofClaudin-5 siRNA. Extravasation of Hoechst H33342 from the brainmicrovessels was manifested by distinct staining of nuclei insurrounding neural and glial cells 24 hours and 48 hours post deliveryof claudin-5 siRNA when compared to control groups. This extravasationwas not evident in sections 72 hours or 1 week post-injection of siRNAtargeting claudin-5. No extravasation of FD-4 was observed in the brainparenchymal tissue at any time point following siRNA injection, or inthe control groups. This highlights the size-selective nature ofRNAi-mediated targeting of claudin-5. Scale bar approx. 20 μm (FIG. 6A).

Extravasation of Hoechst was also evident in 12 μm retinal cryosections,with the Inner Nuclear Layer (INL) appearing stained at 24 hours anddistinct Outer Nuclear Layer (ONL) staining at 48 hours post delivery ofCLDN5 siRNA. In all control groups, Hoechst staining was manifestedsolely in the nuclei of retinal blood vessels which diffuse within theretina as far as the Outer Plexiform Layer (OPL). Scale bar approx. 20μm. (IPL) Inner Plexiform Layer; (GCL) Ganglion Cell Layer (FIG. 6B).

FIG. 7A shows the results of an MRI Scan post injection to assess theblood brain barrier integrity in vivo. The magnetic resonance imaging(MRI) contrasting agent Gd-DTPA was used to ascertain BBB integrity inmice following ablation of claudin-5 transcripts compared to the controlgroups—un-injected control, PBS injected control and non-targeting(Rhodopsin) siRNA injected control mice. The image to the left of thefigure is the contrasting of the mouse brain before injection ofGd-DTPA, while the image to the right is the contrasting of the mousebrain following injection of Gd-DTPA. The images are taken coronallymoving from the ventral aspect of the brain to the dorsal aspect (Lowerimages), with intervening images showing contrasting within thehippocampal and cortex regions. At 24 & 48 hours post-injection ofclaudin-5 siRNA, it was observed that Gd-DTPA crossed the BBB and wasdeposited within the brain. Strong contrasting was also observed in theeye when compared to the control groups of animals at the 48 hour timepoint but not at the other time points. The most significantinfiltration and deposition of Gd-DTPA (742 Da) into the parenchymaoccurred at 24 and 48 hours post-injection of siRNA targeting claudin-5(All

MRI scans were repeated a minimum of twice). This infiltration of thecontrasting agent was not present in the control groups of mice, nor wasit present in mice 72 hours or 1 week post-injection of siRNA targetingclaudin-5.

FIG. 7B shows the results of densitometric analysis of MRI imaging inmouse brain following systemic administration of siRNA targetingclaudin-5. Densitometric analyses of MRI scans in selected regions ofthe Cerebellum, Hippocampus and Cortex for each time point and with eachtreatment were combined and are represented as a bar chart in FIG. 7B.There was a significant increase in contrasting within these regions at24 hours (**P<0.05) and 48 hours (**P<0.05) post injection of claudin-5siRNA when compared to the control groups.

FIG. 7C shows the results of quantitative MRI imaging. The image in FIG.7C is represented as follows; the red end denotes very little change inthe slope of the linear fit, determined for every pixel in the MRI scansof mice. The green areas show some change and blue areas denote a largechange in the rate of Gd-DTPA deposition. The graph below thequantitative image in FIG. 7C shows the change in intensities in leftventricle over a 28-minute timecourse after Gd-DTPA injection. The datain the graph is plotted as the natural logarithm (In) of the signalintensity (y-axis) against time in minutes on the x-axis (each unit onthe x-axis is 128 seconds long). The red line represents thenon-targeting control siRNA injected mouse; the yellow line representsthe 24 hours time point post-injection of siRNA targeting claudin-5;while the green line represents the 48 hour time point post-injection ofclaudin-5 siRNA. Thin lines=raw data of intensities at the 14 timepoints; Thick lines=mathematically calculated linear fit for the timepoints; Dotted lines=the standard error for the linear fit usingchi-squared evaluation.

FIG. 8 shows the results of the administration of 20 mg/kg of TRH tomice following ablation of claudin-5 protein. This graph outlines thedistinct changes in mobility observed upon administration of 20 mg/kgTRH in mice 48 hours after tail vein injection of a non-targeting siRNAand 48 hours post-injection of siRNA targeting claudin-5. When the BBBwas compromised, the behavioural output following TRH injection 48 hourspost delivery of siRNA targeting claudin-5 was manifested by asignificant cessation of mobility that remains for up 5 times longerthan that observed in the non-targeting control mice (P=0.0041).

FIG. 9 shows endothelial cell morphology in liver cryosections. 12 μmcryosections of mouse liver were prepared following injection of siRNAtargeting claudin-5 and using the appropriate control groups. Thebrown/red-rose chromogenic staining in the sections represents theGriffonia simplicifolia-isolectin B4 binding in liver microvasculatureand specifically the endothelial cells lining this microvasculature. Inall sections and all treatments, the microvasculature of the liverappears similar and un-disrupted. Sections were counterstained withHematoxylin.

FIG. 10 shows endothelial cell morphology in lung cryosections.Cryosections of mouse lung were stained with HRP-conjugated Griffoniasimplicifolia-isolectin B4. It is clear that the lung tissue is highlyperfused with microvessels; however the morphology of these vesselsremains un-changed in all experimental groups and at all time pointspost-injection.

Sections were counterstained with Hematoxylin.

FIG. 11 shows endothelial cell morphology in kidney cryosections. Mousekidney cryosections were prepared following injection of siRNA andemploying the appropriate control groups and subsequently stained withHRP-conjugated Griffonia simplicifolia-isolectin B4. Brown/red-rosestaining showed intact kidney microvessels in all treatments and at alltime points post siRNA injection. Sections were counterstained withHematoxylin.

FIG. 12 shows endothelial cell morphology in heart cryosection. Mousehearts were dissected following delivery of siRNA at 24, 48, 72 hoursand 1 week, and using appropriate controls. 12 μm sections were preparedand following staining with Griffonia simplicifolia-isolectin B4, heartassociated microvessels showed similar morphology at all time points andwith all siRNA treatments. Sections were counterstained withHematoxylin.

FIG. 13 shows immunohistochemical analysis of occludin expression inbrain cryosections. Immunohistochemical analysis of occludin expressionand localisation in the microvessels of the brain revealed a continuousand distinct pattern of staining in the microvasculature of all mice andat all time points (Red; Alexa 568=Occludin; Blue-DAPI=nuclei).

FIG. 14 shows the results of Claudin-5 suppression in the retinafollowing siRNA injection. Western blot analysis of claudin-5 expressionin retinal protein lysates showed decreased expression 48 hours posthydrodynamic tail vein injection of siRNA directed against claudin-5.Levels of expression were observed to return to levels similar toun-injected, PBS-injected and non-targeting siRNA injected mice.

FIG. 15 shows the results of Claudin-5 expression in retinal flatmounts.Immunohistochemical analysis of claudin-5 expression in retinalflatmounts from mice receiving a non-targeting siRNA, and mice 24, 48and 72 hours post claudin-5 siRNA injection showed a decreasedlocalisation of claudin-5 at the periphery of endothelial cells liningthe retinal microvessels. This decreased expression of claudin-5 wasconcomitant with increased retinal microvessel permeability.

FIG. 16 shows the results of MRI analysis of Gd-DTPA diffusion acrossthe iBRB. Following contrast enhanced MRI-analysis; it was evident thatthe iBRB was compromised in mice 48 hours post-injection of siRNAtargeting claudin-5. This manifested as increased contrasting within thevitreous of the eye as Gd-DTPA passed from the vasculature to theextravascular spaces.

FIG. 17 shows the results of retinal flatmounts following perfusion ofmice with Hoechst 33352 (562 Da). Following perfusion of mice withHoechst 33352, retinas were dissected out and flatmounted. Hoechst 33352was shown to stain extravascular nuclei in the retinas of mice 24 and 48hours post hydrodynamic tail vein injection of siRNA targeting claudin-5when compared to un-injected mice, mice receiving PBS alone or micereceiving a hydrodynamic tail vein injection of a non-targeting siRNA.

FIG. 18 shows ERG results after GTP injections in IMPDH−/− mice. Rodresponses in a wild-type C-57 mouse were observed to be approximately793 uV in both the left eye and the right eye. In an 11 month oldIMPDH−/− however, the rod responses were observed to be 50.8 uV and 2.48uV in the right eye and left eye respectively. Following suppression ofclaudin-5 however, and injection of GTP at the point of doing asubsequent ERG, the rod tracings were shown to increase significantly,giving b-waves of 193 uV and 121 uV respectively for the right and lefteyes. This increase in rod response were observed in a further 3IMPDH−/− mice post-suppression of claudin-5 and injection of GTP.

FIG. 19 shows the results of Western blot analysis of occludinexpression following hydrodynamic tail vein delivery of occludin siRNA.Levels of expression of occludin were shown to decrease 24 hours postinjection of occludin siRNA (4) and to a lesser extent with occludinsiRNA (2). However, 48 hours post injection of occludin siRNA (1) andoccludin siRNA (2), levels of occludin expression were significantlydecreased compared to mice receiving an injection of a non-targetingsiRNA.

FIG. 20 shows the results of Occludin immunohistochemistry followinghydrodynamic delivery of occludin siRNA. The continuous pattern ofstaining of occludin in the brain microvasculature was observed to bedisrupted 24 hours post-injection of occludin siRNAs. This discontinuouspattern of staining was also evident at the 48 hour time-point foroccludin siRNAs (1) and (2). However the pattern of staining 48 hourspost-injection of occludin siRNAs (3) and (4) had returned to levelssimilar to those observed in the non-targeting controls.

FIG. 21 shows the results of Albumin immunohistochemistry followingsuppression of occluding. Immunohistochemical analysis of albumin inbrain vibratome sections revealed extravascular albumin 24 hourspost-injection of siRNAs numbered (3) and (4). This suggests that 24hours post siRNA injection, the paracellular pathway has beencompromised enough to allow for the passage of molecules up to 70 kDacross the BBB. Blue staining with Hoechst perfusion gives evidence forBBB compromise to a molecule of 562 Da.

FIG. 22 shows the results of immunoglobulin staining in brain vibratomesections following suppression of occluding. Staining of mouse brainsections for mouse immunoglobulins following suppression of occludinrevealed no passage of IgG's into the brain. Mouse IgGs have a molecularweight of approximately 150000 Da and it is clear that they are stillexcluded from the brain when occludin is suppressed.

FIG. 23 shows the results of Western blot analysis of claudin-1expression following hydrodynamic tail vein delivery of claudin-1 siRNA.Following hydrodynamic tail vein delivery of siRNA targeting claudin-1,it was observed that 24 hours post-injection, levels of claudin-1 weredecreased when using siRNAs (2) and (4). This suppression was onlyevident in claudin-1 siRNA (4) at the 48 hour time point post-injectionand although not evident at the 24 hour time-point for claudin-1 siRNA(1), this particular siRNA did significantly decrease claudin-1expression 48 hours post injection.

FIG. 24 shows the results of Hoechst (562 Da) and FD-4 (4,400 Da)perfusion 24 and 48 hours post hydrodynamic tail vein delivery ofclaudin-1 siRNA. Following delivery of a range of siRNAs targetingclaudin-1, a mixture of Hoechst and FD-4 were perfused in mice andvibratome sections of the brains were prepared. It was evident that inall claudin-1 siRNAs used, there was evidence of diffusion of Hoechstfrom the microvessels of the brain as the surrounding neuronal cellswere clearly fluorescing blue when compared to the non-targeting controlmice at the same time points. In all cases, FD-4 was observed to remainwithin the vessels.

FIG. 25 shows the position of one of the Claudin-5 siRNA used in theexperiments (siRNA anti-sense sequence—5′-AGACCCAGAAUUUCCAACGUU-3′corresponding to SEQ ID No. 2), in the Mus musculus Claudin-5 mRNA. Thetarget sequence for this siRNA in the Mus musculus Claudin-5 mRNAdiffers from 5′-AACGTTGGAAATTCTGGGTCT-3′ (SEQ ID NO: 43) in that the AAon the 5′ end is replaced by AG in the mus musculus Claudin-5 mRNA.

FIG. 26 shows T1-weighted MRI images of the Hippocampal region of themouse brain 48 hours post-delivery of siRNA targeting claudin-5 clearlyshows enhanced contrasting within the brain as Gd-DTPA extravasates frombrain microvessels. Gd-DTPA has a molecular weight of 742 Daltons, andits permeation into the brain was only observed at 24 and 48 hours postdelivery of siRNA.

FIG. 27 shows MRI information related to blood flow/volume changeswithin the brains of mice 24 and 48 hours post-high volume tail veininjection of siRNA targeting claudin-5. This data gives information ontwo things, the mean transit time (MTT) and capillary transit time(CTT). The MTT represents the time taken for the labelled spins totravel from the labelling plane (carotid artery—1 cm from imaging slice)to the imaging slice.

FIG. 28 shows the theoretical model for cerebral blood flow and cerebralblood volume fitted to the experimental data for each experimental grouptested group. These are almost exactly the same for each group whichagrees with the findings of the histograms presented in FIG. 27.

FIG. 29 shows the B-values (x-axis) plotted above with MRI signalintensity (y-axis) show no change in the rate of water diffusion in thebrains of mice at 24 and 48 hours post injection of a non-targetingsiRNA or siRNA targeting claudin-5. This constant rate of waterdiffusion from the brain to the blood suggests that the transient BBBopening in itself does not have any profound impact on water diffusionin the brains of mice.

FIG. 30 shows the results after hydrodynamic tail vein injection ofsiRNA targeting claudin-12. The pattern of claudin-12 staining wasobserved to be associated with the brain microvasculature 48 hourspost-injection of a non-targeting control siRNA. However, 48 hourspost-injection of siRNA targeting claudin-12, levels of expression atthe microvessels of the brain were shown to be decreased in both siRNAstested (i.e., CLDN12 siRNA (3) and CLDN12 siRNA (4)).

FIG. 31 shows the results following hydrodynamic tail vein injection ofa non-targeting siRNA or siRNA targeting claudin-12. Mice were perfusedthrough the left ventricle with a solution containing FITC-dextran-4 andHoechst 33342 (562 Da). It was observed that following injection ofsiRNA targeting claudin-12, there was extravasation of Hoechst from themicrovasculature as evidenced by staining of the extravascular nuclei.FD-4 was observed in the microvessels and in both the non-targetingsiRNA and following injection of siRNA targeting claudin-12.

EXAMPLES Example 1: In Vivo Suppression of Claudin-5 Expression at theBlood Brain Barrier of C57/Bl-6 Mice Using Systemic Hydrodynamic TailVein Delivery of siRNA Targeting Claudin-5

Materials

Web-Based siRNA Design Protocols Targeting Claudin-5

siRNAs were selected targeting conserved regions of the published cDNAsequences. To do this, cDNA sequences from mouse were aligned for theClaudin-5 gene and regions of perfect homology subjected to updatedweb-based protocols (Dharmacon, Ambion, Genescript) originally derivedfrom criteria as outlined by Reynolds et al., (2004). Sequences of theclaudin-5 siRNA used in this study were as follows:

(SEQ ID NO. 1) Sense sequence: CGUUGGAAAUUCUGGGUCUUU (SEQ ID NO. 2)Antisense sequence: AGACCCAGAAUUUCCAACGUU

Non-targeting control siRNA targeting human rhodopsin was used as anon-targeting control since rhodopsin is only expressed in photoreceptorcells in the retina and at low levels in the pineal gland of the brain(O'Reilly, M et al., 2007):

(SEQ ID NO. 44) Sense sequence: CGCUCAAGCCGGAGGUCAA (SEQ ID NO. 45)Antisense sequence: UUGACCUCCGGCUUGAGCG

Protocol

In Vivo Delivery of siRNA to Murine BBB by Large Volume HydrodynamicInjection and Subsequent RNA and Protein Analyses

Rapid high pressure, high volume tail vein injections were carried out(Kiang et al., 2005). Wild type C57/Bl6 mice of weight 20-30 g wereindividually restrained inside a 60-ml volume plastic tube. Theprotruding tail was warmed for 5 minutes prior to injection under a 60-Wlamp and the tail vein clearly visualized by illumination from below. 20micrograms of targeting siRNA, or non-targeting siRNA made up with PBSto a volume in mis of 10% of the body weight in grams or PBS alone, wasinjected into the tail vein at a rate of 1 ml/sec using a 26-gauge (26 G3/8) needle. After 24, 48, 72 hours and 1 week, protein was isolatedfrom total brain tissue by crushing brains to a fine powder in liquid N₂and subsequently using lysis buffer containing 62.5 mM Tris, 2% SDS, 10mM Dithiothreitol, 10 μl protease inhibitor cocktail/100 ml (SigmaAldrich, Ireland). The homogenate was centrifuged at 10,000 g for 20mins @ 4° C., and the supernatant was removed for claudin-5 analysis.

Briefly, protein samples were separated on 12% SDS-PAGE gels andtransferred to nitrocellulose membrane overnight using a wet electroblotapparatus. Efficiency of protein transfer was determined using Ponceau-Ssolution (Sigma Aldrich, Ireland). Non-specific binding sites wereblocked by incubating the membrane at room temperature with 5% non-fatdry skimmed milk in Tris-buffered saline (TBS) (0.05 M Tris, 150 mMNaCl, pH 7.5) for 2 hours. Membranes were briefly washed with TBS, andincubated with polyclonal rabbit anti-claudin-5 (Zymed Laboratories, SanFrancisco, Calif.) (1:500) or polyclonal rabbit anti-β-actin (Abcam,Cambridge, UK) (1:1000). Antibodies were incubated with membranesovernight at 4° C. Membranes were washed with TBS, and incubated with asecondary anti-rabbit (IgG) antibody with Horse-Radish-Peroxidase (HRP)conjugates (1:2500), for 3 hours at room temperature. Immune complexeswere detected using enhanced chemiluminescence (ECL).

At the same time points post-delivery of siRNA total RNA was isolatedfrom brains using Trizol (Invitrogen). RNA was then treated withRNase-free DNase (Promega, Madison, Wis., USA) and then chloroformextracted, isopropanol precipitated, washed with 75% RNA grade ethanoland resuspended in 100 μl RNase-free water.

Real-time RT-PCR analysis

RNA was analyzed by real-time RT-PCR using a Quantitect Sybr Green Kitas outlined by the manufacturer (Qiagen-Xeragon) on a LightCycler (RocheDiagnostics, Lewes, UK) under the following conditions: 50° C. for 20min; 95° C. for 15 min; 38 cycles of 94° C. for 15 s, 57° C. for 20 s,72° C. for 10 s.

Primers (Sigma-Genosys. Cambridge, UK) for the seauences amplified wereas follows

CLDN5 (SEQ ID NO. 46) Forward 5′-TTTCTTCTATGCGCAGTTGG-3′ (SEQ ID NO. 47)Reverse 5′-GCAGTTTGGTGCCTACTTCA-3′ β-actin (SEQ ID NO. 48)Forward 5′- TCACCCACACTGTGCCCATCTA-3′ (SEQ ID NO. 49)Reverse 5′-CAGCGGAACCGCTCATTGCCA-3′

cDNA fragments were amplified from claudin-5 and β-actin for each RNAsample a minimum of four times. Results were expressed as a percentageof those from the similarly standardized appropriate control experiment.The reciprocal values compared to the non-targeting control siRNA gavepercentage suppression of claudin-5 expression. Mean values, standarddeviations, and pooled t tests were calculated using GraphPad Prism©.Differences were deemed statistically significant at P<0.05.

Indirect Immunostaining for Claudin-5 Using Confocal Laser ScanningMicroscopy (CLSM) for Analysis

Brain cryosections were blocked with 5% Normal Goat Serum (NGS) in PBSfor 20 mins at room temperature. Primary antibody (Rabbitanti-Claudin-5, Zymed, California) was incubated on sections overnightat 4° C. Following this incubation, sections were washed 3 times in PBSand subsequently blocked again with 5% NGS for 20 mins at roomtemperature. A secondary rabbit IgG-Cy3 antibody was incubated with thesections at 37° C. for 2 hours followed by 3 washes with PBS. Allsections were counterstained with DAPI for 30 seconds at a dilution of1:5000 of a stock 1 mg/ml solution. Analysis of stained sections wasperformed with an Olympus FluoView TM FV1000 Confocal microscope.

Assessment of BBB Integrity by Perfusion of a Biotinylated TracerMolecule

Following RNAi-mediated ablation of transcripts encoding claudin-5, atracer molecule was used to determine the extent to which the TJ's ofthe BBB had been affected. The biotinylated reagent EZ-Link TMSulfo-NHS-Biotin (Pierce) (1 ml/g body weight of 2 mg/ml EZ-Link TMSulfo-NHS-Biotin, 443 Da) was perfused for 5 minutes through the leftventricle of mice 24, 48, 72 hours and 1 week post-hydrodynamic deliveryof claudin-5 siRNA. Following perfusion with the tracer molecule, thewhole brain was dissected and placed in 4% PFA pH 7.4 overnight at 4° C.and subsequently washed 4×15 mins with PBS. Following cryoprotectionwith sucrose, frozen sections were cut on a cryostat at −20° C. andincubated with streptavidin conjugated to the fluorescent probe FITC.This allowed for the assessment of leakage of the biotinylated reagentof molecular weight 443 Da from the microvessels of the brain. Allsections were counterstained with 4′,6-diamidine-2-phenylindole-dihydrochloride (DAPI; Sigma Aldrich,Ireland) for 30 seconds at a dilution of 1:5000 of a stock 1 mg/mlsolution, and sections were visualized using an Olympus FluoView TMFV1000 Confocal microscope.

Assessment of BBB/BRB Permeability to Molecules of 562 Daltons and 4,400Daltons

In order to determine the permeability of brain and retinal microvesselsto a molecule of 562 Daltons, mice were perfused through the leftventricle with 500 μl/g body weight of PBS containing 100 μg/ml Hoechststain H33342 (Sigma Aldrich, Ireland) and 1 mg/ml FITC-Dextran-4 (FD-4)24, 48, 72 hours and 1 week post-hydrodynamic delivery of claudin-5siRNA. Following perfusion, the whole brain was dissected and placed in4% PFA pH 7.4 overnight at 4° C. and subsequently washed 4×15 mins withPBS. Brains were then embedded in 4% agarose and 50 μm sections were cutusing a Vibratome®. Whole eyes were removed and fixed with 4% PFA, andfollowing washing with PBS and cryoprotection using a sucrose gradient,12 μm cryosections were cut using a cryostat. Following analysis ofretinal cryosections with an Olympus FluoView TM FV1000 Confocalmicroscope, images were oriented correctly using Adobe® Photoshop®.

Magnetic Resonance Imaging

Following injection of siRNA and using appropriate controls, BBBintegrity was assessed in vivo via MRI, using a dedicated small rodentBruker BioSpec 70/30 (i.e. 7T, 30 cm bore) with an actively shielded USRMagnet. Mice were anaesthetised with isofluorane, and physiologicallymonitored (ECG, respiration and temperature) and placed on anMRI-compatible support cradle, which has a built-in system formaintaining the animal's body temperature at 37° C. The cradle was thenpositioned within the MRI scanner. Accurate positioning is ensured byacquiring an initial rapid pilot image, which is then used to ensure thecorrect geometry is scanned in all subsequent MRI experiments. Uponinsertion into the MRI scanner, high resolution anatomical images of thebrain were acquired (100 μm in-plane and 500 μm through-plane spatialresolution). BBB integrity was then visualised in high resolution T₁weighted MR images before and after injection of a 0.1 mM/L/kg bolus ofGd-DTPA (Gadolinium diethylene-triamine pentaacetic acid), administeredvia the tail vein.

Electroretinographic Analysis of IMPDH−/− Mice and GTP Injection

IMPDH−/− mice that had received a hydrodynamic injection of siRNAtargeting claudin-5 were dark-adapted overnight and prepared forelectroretinography under dim red light. Pupillary dilation was carriedout by installation of 1% cyclopentalate and 2.5% phenylephrine. Animalswere anesthetized by intraperitoneal injection of ketamine (2.08 mg per15 g body weight) and xylazine (0.21 mg per 15 g body weight). Once theanimal was anaesthetized, GTP was injected intraperitoneally. The ERGcommenced ten minutes after administration of anesthetic. Standardisedflashes of light were presented to the mouse in a Ganzfeld bowl toensure uniform retinal illumination. The ERG responses were recordedsimultaneously from both eyes by means of gold wire electrodes (RolandConsulting Gmbh) using Vidisic (Dr Mann Pharma, Germany) as a conductingagent and to maintain corneal hydration. The eye was maintained in aproptosed position throughout the examination by means of a smallplastic band placed behind the globe. Reference and ground electrodeswere positioned subcutaneously, approximately 1 mm from the temporalcanthus and anterior to the tail respectively. Body temperature wasmaintained at 37° C. using a heating device controlled by a rectaltemperature probe. Responses were analysed using a RetiScan RetiPortelectrophysiology unit (Roland Consulting Gmbh). The protocol was basedon that approved by the International Clinical Standards Committee forhuman electroretinography.

Immunohistochemical Analysis of Flatmounted Retinas

Whole eyes were fixed for 4 hours in 4% paraformaldehyde followed by 3washes with phosphate buffered saline (PBS). Retinas were dissected outof the eyes and blocked/permeabilised by incubation with PBS containing0.5% Triton X-100 and 5% normal goat serum (NGS). Retinas weresubsequently incubated overnight in permeabilisation buffer containing1% NGS and a 1:50 dilution of Rabbit anti-claudin-5 antibody (Zymed).Following 10 washes with PBS over a period of 2 hours, retinas wereincubated for 6 hours at room temperature with a rabbit IgG antibodyconjugated with the fluorescent probe Cy-3. Following 10 washes with PBSover a period of 2 hours, retinas were flatmounted and viewed using aconfocal microscope.

Endothelial Cell Morphology of Major Organs

Following hydrodynamic injection of siRNA targeting claudin-5,cryosections were prepared of all the major organs, the heart, liver,lung and kidney. Sections were incubated overnight at 4° C. withHRP-conjugated Griffonia simplicifolia-isolectin B4 in order to stainthe endothelial cells of organs.

Results

Hydrodynamic Tail Vein Injection of CLDN5 siRNA Attenuates Claudin-5Expression

Following delivery of 20 μg claudin-5 siRNA, mice were left for 24, 48,72 hours and 1 week, after which time brains were dissected and proteinand RNA isolated as described previously. After 24 hours, levels ofexpression of Claudin-5 were markedly decreased when compared to Control(Un-injected), PBS injected and Non-targeting (Rhodopsin) controls. Miceinjected with CLDN5 siRNA and subsequently left for 48 hours also showedsignificant decreases in Claudin-5 expression when compared to thecontrols employed. At 72 hours post-injection, this observed decrease inClaudin-5 expression was less evident, and levels of expression appearedsimilar to those observed in the control groups. One week post injectionof claudin-5 siRNA, levels of expression of claudin-5 were similar tothose in the control groups of animals. All blots are representative ofat least 3 separate experiments (FIG. 1A).

Levels of claudin-5 mRNA were determined by RT-PCR analysis and showed asignificant decrease 24 hours post-injection of siRNA targetingclaudin-5. This highly significant decrease was not observed at thelater time points, and showed claudin-5 mRNA levels suppressed up to 95%with respect to the non-targeting control siRNA (FIG. 1B). Levels ofclaudin-5 mRNA 48 hours, 72 hours and 1 week post injection were similarto those observed in the control groups (FIG. 1B).

Claudin-5 Expression and Localisation Becomes Altered in Brain CapillaryEndothelial Cells Following Injection of Claudin-5 siRNA

The level of expression of claudin-5 at the TJ in brain capillaryendothelial cells changed dramatically following suppression ofclaudin-5 expression. As shown in FIG. 2, immunohistochemical analysisof claudin-5 expression and localisation in the microvessels of thebrain which revealed a linear and distinct pattern of staining at theperiphery of endothelial cells of the BBB in the un-injected, PBSinjected and non-targeting control (Rhodopsin) mice at all time pointsafter injection.

Claudin-5 expression was linear and intense at the periphery ofendothelial cells lining the microvessels of the un-injected, PBSinjected and non-targeting siRNA (Rhodopsin) injected control groups.However, 24 hours post injection of claudin-5 siRNA, this stainingpattern appeared less intense, and by 48 hours post-injection there wasa marked decrease in the presence of claudin-5 expression at theperiphery of brain capillary endothelial cells throughout the brain whencompared to un-injected, PBS-injected or non-targeting siRNA injectedmice. At 72 hours post-injection, claudin-5 expression was stillattenuated, yet a linear pattern of staining was evident in thecryosections (FIG. 2).

These results are representative of at least 5 separate experiments

Claudin-5 siRNA Causes Increased Permeability at the BBB

FIG. 5 shows the results of the assessment of the blood brain barrierintegrity by perfusion of a biotinylated tracer molecule. EZ-Link TMSulfo-NHS-Biotin was perfused through the left ventricle in micefollowing exposure to experimental conditions. Upon incubation ofcryosections with streptavidin conjugated to the fluorescent probe FITC,the integrity of the BBB was observed as green fluorescence within themicrovessels in the control groups (un-injected, PBS injected and anon-targeting siRNA. However, 24 hours post-injection of siRNA targetingclaudin-5, it was observed that the fluorescence detected was diffuseand outside of the microvessels in contrast to the control groups at thesame time point. At 48 hours post-injection of claudin-5 siRNA, thedistribution of the biotinylated molecule was abundant in the brainparenchyma, while this permeability was still evident 72 hourspost-delivery of siRNA when compared to the control groups at the sametime points. In mice 1 week post-injection of siRNA targeting claudin-5,it was observed that the primary amine-reactive biotinylated reagent didnot deposit in the parenchyma following perfusion for 5 minutes. TheEZ-Link TM Sulfo-NHS-Biotin was observed to remain in the microvesselsof the brain due to an intact BBB.

All tracer experiments were repeated in mice at least 5 times.

In summary, upon delivery of siRNA targeting claudin-5 to braincapillary endothelial cells, an increase in permeability to a smallbiotinylated molecule (443 Da) was observed after 24 hours. The passageof this molecule across the BBB became very distinct 48 hourspost-injection, with large quantities of EZ-Link TM Sulfo-NHS-Biotininfiltrating the parenchymal tissue of the brain. The passage of thismolecule across the BBB was still evident 72 hours post-injection ofclaudin-5 siRNA, however 1 week post-injection, there was no evidencefor BBB compromise and the EZ-Link TM Sulfo-NHS-Biotin was shown toremain within the microvessels of the brain (FIG. 5).

FIGS. 6A, and 6B show the results of the assessment of the blood brainbarrier integrity by perfusion of a biotinylated tracer molecule at ahigh magnification. It was observed that upon perfusion of the primaryamine reactive biotinylated reagent (443 Da) for 5 minutes 24 hourspost-injection of siRNA targeting claudin-5 caused an infiltration ofthe molecule into the parenchyma of the brain when compared to theun-injected, PBS injected and non-targeting siRNA injected mice. Thisinfiltration was detected in abundance 48 hours post-delivery of siRNA,and was still evidenced up to and including 72 hours following injectionof claudin-5 siRNA. In mice 1 week post-injection of claudin-5 siRNA, itwas observed that the

EZ-Link TM Sulfo-NHS-biotin remained in the microvessels of the brainand failed to deposit in the parenchyma.

In summary, upon analysis of brain cryosections at a highermagnification in the dentate gyrus region of the hippocampus (for easeof recognition), it was apparent that 1 week post-injection of claudin-5siRNA, the BBB did not allow for the passage of EZ-Link TMSulfo-NHS-Biotin that was so clearly evidenced 48 hours post-injectionof siRNA (FIGS. 6A and 6B).

Claudin-5 siRNA Causes Increased Permeability at the BBB and BRB to aMolecule of 562 Daltons

Intriguingly, upon perfusion of the nuclear stain Hoechst H33342 (562Daltons) and the FITC labelled dextran, FD-4 (4,400 Daltons),extravasation of Hoechsct was observed up to and including 48 hourspost-delivery of siRNA targeting claudin-5, however, unlike EZ-Link TMSulfo-NHS-Biotin, this extravasation was not evident 72 hours post siRNAdelivery, suggesting a restoration of barrier integrity to a molecule of562 Daltons, and implying a time dependent and size-selective opening ofthe BBB. Hoechst H33342 dye extravasation from the brain microvesselswas manifested by nuclear staining of surrounding neural and glial cellsin the parenchyma. FD-4 remained within the microvessels of the brainvasculature and no extravasation was evident at any time pointpost-injection of siRNA (FIG. 6B).

Moreover, upon analysis of retinal cryosections, we observed thatHoechst H33342 extravasated from the retinal microvessels, staining theInner Nuclear Layer (INL) and Outer Nuclear Layer (ONL) of the retina upto 48 hour post-delivery of siRNA targeting claudin-5 (FIG. 6A).

MRI Analysis Showed Impairment of BBB Integrity 48 Hours Post-Injectionof Claudin-5 siRNA

FIG. 7A shows the results of an MRI Scan post injection to assess theblood brain barrier integrity in vivo. The magnetic resonance imaging(MRI) contrasting agent Gd-DTPA was used to ascertain BBB integrity inmice following ablation of claudin-5 transcripts. At 48 hourspost-injection of claudin-5 siRNA, it was observed that Gd-DTPA crossedthe BBB and was deposited in the parenchymal tissue of the brain. Theimage to the left of the figure is the contrasting of the mouse brainbefore injection of Gd-DTPA, while the image to the right is thecontrasting of the mouse brain following injection of Gd-DTPA. Theimages are taken coronally moving from the ventral aspect of the brainto the dorsal aspect, and reveal significant deposition of Gd-DTPA (742Da) in the parenchyma 48 hours post-injection of siRNA targetingclaudin-5.

This infiltration of the contrasting agent was not present in theun-injected, PBS injected or non-targeting siRNA injected mice, nor wasit present in mice 72 hours or 1 week post-injection of siRNA targetingclaudin-5.

In summary, infiltration of Gd-DTPA into the brain parenchymal tissuewas observed as widespread and intense contrasting throughout the brainwhen compared to un-injected,

PBS injected and non-targeting siRNA injected mice, indicating that theBBB was compromised enough to allow for the passage of a molecule of 742Da in size. MRI scans on mice 72 hours and 1 week post-injection ofclaudin-5 siRNA revealed an intact barrier with no deposition of Gd-DTPAin the parenchymal tissue, highlighting this BBB disruption as atransient event (FIG. 7A).

CONCLUSION

As shown in Example 1, the hydrodynamic approach for delivery of siRNA'sto endothelial cells of the brain microvasculature is highly efficientin suppressing claudin-5 expression (FIGS. 1A and 1B). This method ofdelivery caused little harm and was well tolerated in mice. The Westerndata showed that maximum suppression of claudin-5 was achieved 48 hoursafter delivery of the siRNA, with levels of expression of claudin-5returning to normal between 72 hours and 1 week after injection. Thus,the reversible RNAi-mediated opening of the BBB using siRNA targetingclaudin-5 is now possible.

It was then determined whether similar to the claudin-5 knockout mouse,the BBB became compromised to small molecules when claudin-5 expressionwas suppressed. At the periphery of endothelial cells in the brainmicrovasculature, levels of claudin-5 appeared strong and “linear-like”upon immunohistochemical analysis of all the control groups employed.However, when claudin-5 was targeted, this linear appearance ofexpression became discontinuous and fragmented, with levels appearingdramatically reduced 48 hours after injection of claudin-5 siRNA (FIG.2). Moreover, upon perfusion of mice with the biotinylated moleculeEZ-Link TM Sulfo-NHS-Biotin for 5 minutes, a significant compromise inbarrier function was observed up to and including 72 hours post deliveryof siRNA targeting claudin-5. EZ-Link TM Sulfo-NHS-Biotin has amolecular weight of 443 Da, and will normally not cross the BBB if theTJ's are intact as observed in the control groups. Interestingly, 1 weekafter delivery of claudin-5 siRNA, this molecule no longer crossed theBBB, suggesting that consistent with Real-Time PCR and Western analyses,this compromise in BBB function is a transient and reversible process.

During this study, no distinct or noticeable behavioural changes inthese mice, while the gross histology of both vibratome and cryosectionsof the brain appeared normal under all experimental conditions.

Similar to claudin-5 knockout mouse, the MRI contrasting agent Gd-DTPAwas also found to cross the BBB and deposit in the parenchymal tissue ofthe brain post siRNA injection. In fact extremely large quantities ofGd-DTPA were deposited in the brain 48 hours post delivery of claudin-5siRNA. This BBB breakage to a molecule of 742 Da was a transient event,as 72 hours and 1 week post injection of siRNA targeting claudin-5,there appeared to be no deposition of Gd-DTPA. The significance of theseresults was that as well as being a transient event, suppression ofclaudin-5 appeared to be causing a size-selective change in thepermeability of the barrier, as evidenced from the observation thatwhile a molecule of 443 Da crossed the BBB 72 hours post delivery ofsiRNA, a molecule of 742 Da failed to do so at the same time point (FIG.7A), and a molecule of 562 Da crossed the BBB at 24 and 48 hours postdelivery of siRNA while a molecule of 4,400 Da failed to do so (FIGS. 6Aand 6B).

As siRNA was administered via the tail vein, and given the fact thatclaudin-5 is expressed in microvascular endothelial cells of the lungand the heart, we wished to assess whether siRNA targeting claudin-5would adversely affect endothelial cell morphology in the liver, lung,kidney or heart. Cryosections of each of these organs were prepared atall time points following injection of siRNA targeting claudin-5 andincorporating the appropriate controls. Sections were stained withHRP-conjugated Griffonia simplicifolia-isolectin B4, which binds tointact endothelial cells, and showed that endothelial cell morphologyappeared similar at all time points and in all major organs followingsiRNA injection when compared to the control groups (FIGS. 9-12). Therole of claudin-5 in organs other than the brain and eye has not beenwell characterised, and it is important to note that it does not appearto be fundamental in maintaining the size-selective properties of thetight junctions associated with these other organs.

In conclusion, it is now possible to systemically deliver siRNAmolecules to the endothelial cells of the BBB and BRB. Targetedsuppression of the TJ protein claudin-5 causes both a transient andsize-selective increase in paracellular permeability of the barrier,which may allow for the delivery of molecules which would otherwise beexcluded from the brain.

Example 2: Delivery of Thyrotropin Releasing Hormone (TRH) Across theBlood Brain Barrier (BBB) to Claudin-5 Suppressed Mice

Materials and Methods

Thyrotropin Releasing Hormone (TRH) (Sigma Aldrich, Ireland)

TRH has been proposed as having distinct neuroprotective effects. Italso induces “wet dog shake” behavioural outputs when administered torats. However, TRH has several disadvantages, including its instabilityand resulting short duration of action and its slow permeation acrossthe BBB.

Delivery of TRH to Claudin-5 Suppressed Mice

The protocol of Example 1 was followed to produce transiently claudin-5suppressed mice.

48 hours post-delivery of siRNA targeting claudin-5 or a non-targetingsiRNA, a 200 μl of a solution containing 20 mg/kg Thyrotropin ReleasingHormone (TRH) was injected to the claudin-5 suppressed mice. TRH wasinjected in the tail vein and immediately, the behavioural output ofmice was assessed by filming them in a clear Perspex box.

RESULTS AND CONCLUSION

As shown in FIG. 8, following ablation of claudin-5 protein, a distinctincrease in the length of time C57/Bl6 mice remain immobile uponadministration of 20 mg/kg TRH was observed.

This behavioural output was significantly different from the behaviourobserved in the non-targeting control mice, and clearly suggested thatdelivery of TRH was significantly enhanced when the BBB was compromised.

These results show that the protocol of Example 1 can be used to openthe BBB to allow delivery of compositions to the BBB which previouslywould not have been possible. These results clearly suggest thatdelivery of TRH (359.5 Da) was significantly enhanced when the BBB wasreversibly, transiently opened in a controlled size selective manner.

Example 3

In Vivo Suppression of Claudin-1 Expression at the Blood Brain Barrierof C57/Bl-6 Mice Using Systemic Hydrodynamic Tail Vein Delivery of siRNATargeting Claudin-1

Materials

Web-based siRNA design protocols targeting Claudin-1

CLDN1 (1) target sequence: GCAAAGCACCGGGCAGAUA (SEQ ID NO. 32):(SEQ ID NO. 9) Sense sequence: AUAGACGGGCCACGAAACGUU (SEQ ID NO. 10)Anti-sense strand: CGUUUCGUGGCCCGUCUAUUUCLDN1 (2) target sequence: GAACAGUACUUUGCAGGCA: (SEQ ID NO. 33)(SEQ ID NO. 11) Sense strand: ACGGACGUUUCAUGACAAGUU (SEQ ID NO. 12)Anti-sense strand: CUUGUCAUGAAACGUCCGUUUCLDN1 (4) target sequence: UUUCAGGUCUGGCGACAUU (SEQ ID NO. 34):(SEQ ID NO. 13) Sense sequence: UUACAGCGGUCUGGACUUUUU (SEQ ID NO. 14)Anti-sense strand: AAAGUCCAGACCGCUGUAAUU

Methods

The protocols used were identical to the protocols used in Example 1.

RESULTS AND CONCLUSIONS

Results are shown in FIGS. 3A, 23 and 24.

This example shows that siRNA directed against claudin-1 causes anincrease in paracellular permeability at the BBB to a molecule of 562Daltons but not 4,400 Daltons. Suppression of claudin-1 appears to causea size-selective opening of the BBB in a manner similar to that observedwhen claudin-5 was suppressed.

Example 4

In Vivo Suppression of Occludin Expression at the Blood Brain Barrier ofC57/BL-6 Mice Using Systemic Hydrodynamic Tail Vein Delivery of siRNATargeting Occludin

Materials

Web-Based siRNA Design Protocols Targeting Occludin

Occl (1) target sequence: GUUAUAAGAUCUGGAAUGU (SEQ ID NO. 35):(SEQ ID NO. 15) Sense sequence: UGUAAGGUCUAGAAUAUUGUU (SEQ ID NO. 16)Anti-sense sequence: CAAUAUUCUAGACCUUACAUUOccl (2) target sequence: GAUAUUACUUGAUCGUGAU (SEQ ID NO. 36):(SEQ ID NO. 17) Sense sequence: UAGUGCUAGUUCAUUAUAGUU (SEQ ID NO. 18)Anti-sense sequence: CUAUAAUGAACUAGCACUAUUOccl (3) target sequence: CAAAUUAUCGCACAUCAAG (SEQ ID NO. 37):(SEQ ID NO. 19) Sense sequence: GAACUACACGCUAUUAAACUU (SEQ ID NO. 20)Anti-sense sequence: GUUUAAUAGCGUGUAGUUCUUOccl (4) target sequence: AGAUGGAUCGGUAUGAUAA (SEQ ID NO. 38):(SEQ ID NO. 21) Sense sequence: AAUAGUAUGGCUAGGUAGAUU (SEQ ID NO. 22)Anti-sense sequence: UCUACCUAGCCAUACUAUUUU

Methods

The protocols used were identical to the protocols used in Example 1.

RESULTS AND CONCLUSIONS

Results are shown in FIGS. 3B, 19, 20, 21 and 22

This example shows that siRNA directed against Occludin causes anincrease in paracellular permeability at the BBB to molecules greaterthan 70,000 Daltons, as this is the approximate weight of albumin.Suppression of occludin at the BBB does produce a larger size-exclusionlimit however. It was observed that while albumin deposition occurred 24hours post-injection of occludin siRNAs 3 & 4, there was noextravasation of immunoglobulins (IgGs) in the blood. IgG's have anapproximate molecular weight of 120,000 daltons.

Example 5

In Vivo Suppression of Claudin 12 Expression at the Blood Brain Barrierof C57/BL-6 Mice Using Systemic Hydrodynamic Tail Vein Delivery of siRNATargeting Claudin-12

Materials

Web-based siRNA design protocols targeting Claudin 12

CLDN12 SIRNA (3) Target sequence: (SEQ ID NO. 41) GUAACACGGCCUUCAAUUC(SEQ ID No. 28) 5′-GUAACACGGCCUUCAAUUCUU-3′ (SEQ ID No. 29)5′-GAAUUGAAGGCCGUGUUACUU-3′ CLDN12 SIRNA (4) Target sequence:(SEQ ID NO. 42) GGUCUUUACCUUUGACUAU (SEQ ID No. 30)5′-AAUCUUUACCUUUGACUAUUU-3′ (SEQ ID No. 31) 5′-AUAGUCAAAGGUAAAGAUUUU-3′

Methods

The protocols used were identical to the protocols used in Example 1.

RESULTS AND CONCLUSIONS

Claudin-12 levels were shown to decrease following hydrodynamic tailvein injection of siRNA targeting claudin-12 (FIG. 12). The pattern ofclaudin-12 staining was observed to be associated with the brainmicrovasculature 48 hours post-injection of a non-targeting controlsiRNA. However, 48 hours post-injection of siRNA targeting claudin-12,levels of expression at the microvessels of the brain were shown to bedecreased in both siRNAs tested (i.e., CLDN12 siRNA (3) and CLDN12 siRNA(4)).

Following hydrodynamic tail vein injection of a non-targeting siRNA orsiRNA targeting claudin-12, mice were perfused through the leftventricle with a solution containing FITC-dextran-4 and Hoechst 33342(562 Da). It was observed that following injection of siRNA targetingclaudin-12, there was extravasation of Hoechst from the microvasculatureas evidenced by staining of the extravascular nuclei. FD-4 was observedin the microvessels and in both the non-targeting siRNA and followinginjection of siRNA targeting claudin-12 (FIG. 31).

Example 6

Gd-DTPA 48 Hours Post-Delivery of siRNA Targeting Claudin-5

Materials and Methods

The protocol of Example 1 was followed to produce transiently claudin-5suppressed mice. 48 hours post-delivery of siRNA targeting claudin-5 ora non-targeting siRNA, a solution containing Gd-DTPA injected to theclaudin-5 suppressed mice. Gd-DTPA was injected in the tail vein toassess whether Gd-TPA would permeate across the BBB. Following injectionof siRNA and using appropriate controls, BBB integrity to a molecule of742 Daltons (Gd-DTPA) was assessed via MRI, using a dedicated smallrodent Bruker BioSpec 70/30 (i.e. 7T, 30 cm bore) with an activelyshielded USR Magnet. Mice were anaesthetised with isofluorane, andphysiologically monitored (ECG, respiration and temperature) and placedon an MRI-compatible support cradle, which has a built-in system formaintaining the animal's body temperature at 37° C. The cradle was thenpositioned within the MRI scanner. Accurate positioning is ensured byacquiring an initial rapid pilot image, which is then used to ensure thecorrect geometry is scanned in all subsequent MRI experiments. Uponinsertion into the MRI scanner, high resolution anatomical images of thebrain were acquired (100 μm in-plane and 500 μm through-plane spatialresolution). BBB integrity was then visualised in high resolution T₁weighted MR images before and after injection of a 0.1 mM/L/kg bolus ofGd-DTPA (Gadolinium diethylene-triamine pentaacetic acid), administeredvia the tail vein. Following injection of Gd-DTPA, repeated 3 minuteT₁-weighted scans were performed over a period of 30 minutes, and imagesshown are representative of the final scans of this 30 minute period.Statistical analysis of all densitometric results of combined regions ofthe Cerebellum, Hippocampus and Cortex was performed using ANOVA, withsignificance represented by a P value of ≤0.05, and results arepresented both graphically and in a quantitative image depicting therate of Gd-DTPA deposition within the brain. All MRI scans wereperformed on 2 mice from each experimental treatment.

Results

FIG. 26 shows T1-weighted MRI images of the Hippocampal region of themouse brain 48 hours post-delivery of siRNA targeting claudin-5 clearlyshows enhanced contrasting within the brain as Gd-DTPA extravasates frombrain microvessels. Gd-DTPA has a molecular weight of 742 Daltons, andits permeation into the brain was only observed at 24 and 48 hours postdelivery of siRNA.

FIG. 27 shows MRI information related to blood flow/volume changeswithin the brains of mice 24 and 48 hours post-high volume tail veininjection of siRNA targeting claudin-5. This data gives information ontwo things, the mean transit time (MTT) and capillary transit time(CTT). The MTT represents the time taken for the labelled spins totravel from the labelling plane (carotid artery ˜1 cm from imagingslice) to the imaging slice.

This is calculated from the first moment or mean of the curve. From thesecond moment of the curve (variance) we get the CTT, which is the timetaken for the labelled spins to be distributed over the imaging slice byexchange from capillary bed to tissue. With up to 8 animals per group,we are seeing no significant differences in blood flow within the majorvessels in the brain or the capillaries, which is quite promising giventhe high volume of injection required to deliver claudin-5 siRNA tobrain microvascular endothelial cells.

FIG. 28 shows the theoretical model for cerebral blood flow and cerebralblood volume fitted to the experimental data for each experimental grouptested group. These are almost exactly the same for each group whichagrees with the findings of the histograms presented in FIG. 27.

CONCLUSION

In conclusion, these results show that we are not observing anydifference in blood flow or blood volume in the large vessels in thebrain or in the microvasculature and suggests that in cases of cerebraloedema, claudin-5 siRNA may in fact allow for an increased rate waterdiffusion at the site of injury in the brain.

Example 7

Water Diffusion in the Brains of Mice 24 and 48 Hours after ReceivingNon-Targeting or Claudin-5 siRNA

Materials and Methods

The protocol of Example 1 was followed to produce transiently claudin-5suppressed mice. 24 and 48 hours post-delivery of siRNA targetingclaudin-5 or a non-targeting siRNA mice were anaesthetised withisofluorane, and physiologically monitored (ECG, respiration andtemperature) and placed on an MRI-compatible support cradle, which has abuilt-in system for maintaining the animal's body temperature at 37° C.The cradle was then positioned within the MRI scanner. Accuratepositioning is ensured by acquiring an initial rapid pilot image, whichis then used to ensure the correct geometry is scanned in all subsequentMRI experiments. Upon insertion into the MRI scanner, high resolutionanatomical images of the brain were acquired (100 μm in-plane and 500 μmthrough-plane spatial resolution). Water diffusion scans weresubsequently undertaken. Using a standard diffusion imaging sequencesuch as a spin-echo EPI imaging sequence with diffusion gradients(Stejskal-Taner gradients) in order to acquire images over a large rangeof b-values. In this way, the effect of the technique in the vascularcompartment and in the brain parenchymal tissue can be compared. Allexperiments were carried out on a 7T Brüker small-bore system with 400mT/m maximum gradient strength. Image processing and calculation of ADCswas carried out using IDL.

Results FIG. 29 shows the B-values (x-axis) plotted above with MRIsignal intensity (y-axis) show no change in the rate of water diffusionin the brains of mice at 24 and 48 hours post injection of anon-targeting siRNA or siRNA targeting claudin-5. This constant rate ofwater diffusion from the brain to the blood suggests that the transientBBB opening in itself does not have any profound impact on waterdiffusion in the brains of mice.

In conclusion, the results shown in FIG. 30 show the rates of waterdiffusion in the brains of mice 24 and 48 hours after receivingnon-targeting or claudin-5 siRNA. Essentially, there are no changes indiffusion in any mice under these experimental conditions. This isanother important observation and as Example 6 also suggests that incases of cerebral oedema, claudin-5 siRNA may in fact allow for anincreased rate of water diffusion at the site of injury in the brain.

What is claimed:
 1. A method for the treatment of a disease or disorderselected from a neurodegenerative disorder, a neuropsychiatric disorder,brain tumor, and retinal disorder, the method comprising the reversible,transient and controlled RNAi-mediated size selective opening of theparacellular pathway of the blood brain barrier wherein the methodcomprises: identifying a subject at risk for developing the disease ordisorder; administering an effective amount of an RNAi inducing agenttargeting tight junction proteins selected from occludin, claudin 1-19or 21 by delivery of the RNAi inducing agent to result in the transientand reversible RNAi-mediated suppression of blood brain barrier tightjunction protein transcripts in brain capillary endothelial or retinalendothelial cells and allow the permeation of active agents used in thetreatment of the disease or disorder to the brain capillary endothelialand/or retinal cells; and administering an active agent suitable for thetreatment of the disease or disorder.
 2. The method according to claim 1wherein the RNAi agent is: siRNA, shRNA or an RNAi-inducing vector whosepresence within a cell results in production of an siRNA, shRNA ormiRNA.
 3. The method according to claim 1 involving systemic delivery ofthe RNAi inducing agent to the subject.
 4. The method according to claim1 wherein the RNAi inducing agent targeting the tight junction proteinstransiently opens the blood brain barrier to allow delivery of theactive agent across the blood brain barrier and the treatment comprisesthe simultaneous or sequential administration of the active agent andRNAi inducing agent.
 5. The method according to claim 1 wherein a highconcentration of the RNAi inducing agent is delivered to the subject. 6.The method according to claim 1 wherein systemic delivery takes place byhydrodynamic delivery or non-hydrodynamic delivery.
 7. The methodaccording to claim 1 wherein cationic polymers, modified cationicpolymers, peptide molecular transporters, lipids, liposomes,non-cationic polymers and/or viral vectors are used for delivery of theRNAi inducing agent.
 8. The method according to claim 1 whereinmolecules less than approximately 1 kDa permeate across the braincapillary endothelial and/or retinal endothelial cells.
 9. The methodaccording to claim 1 wherein molecules less than approximately 800 Da,permeate across the brain capillary endothelial and/or retinalendothelial cells.
 10. The method according to claim 1 whereinRNAi-mediated suppression commences from approximately 24 hours postdelivery of the RNAi inducing agent and lasts up to approximately 72hours post delivery of the RNAi inducing agent.
 11. The method accordingto claim 1 wherein the RNAi inducing agent is siRNA.
 12. The methodaccording to claim 1 wherein the claudin is selected from claudin 1,claudin-5 and/or claudin-12.
 13. The method according to claim 1 whereinthe claudin is claudin-5.
 14. The method according to claim 1 whereinthe siRNA is selected from any one of SEQ ID Nos 1 and 2; 3 and 4; 5 and6; 7 and 8; 9 and 10; 11 and 12; 13 and 14; 15 and 16; 17 and 18; 19 and20; 21 and 22; 24 and 25; 26 and 27; 28 and 29; or 30 and
 31. 15. Themethod according to claim 1 wherein one or more siRNAs targetingdifferent TJ proteins are used.
 16. The method of claim 1 which allowsthe permeation of active agents less than 15 kDa.